Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof

ABSTRACT

An infectious clone based on the genome of a wild-type RNA virus is produced by the process of providing a host cell not susceptible to infection by the wild-type RNA virus, providing a recombinant nucleic acid based on the genome of the wild-type RNA virus, transfecting the host cell with the recombinant nucleic acid and selecting for infectious clones. The recombinant nucleic acid comprises at least one full-length DNA copy or in vitro-transcribed RNA copy or a derivative of either. The infectious clones can be used in single or dual purpose vaccines and in viral vector vaccines.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of co-pendingapplication U.S. Ser. No.09/874,626, filed Jun. 5, 2001, which is acontinuation of application Ser. No.09/297,535 filed Oct. 12, 1999, nowU.S. Pat. No. 6,268,199, which was the National Stage of InternationalApplication No. PCT/NL97/00593 filed Oct. 29, 1997 (published in Englishon May 7, 1998 as PCT International Publication Number WO 98/18933), thecontents of all of which are incorporated by this reference.

TECHNICAL FIELD

[0002] The invention relates to the field of RNA viruses and infectiousclones obtained from RNA viruses. Furthermore, the invention relates tovaccines and diagnostic assays obtainable by using and modifying suchinfectious clones of RNA viruses.

BACKGROUND

[0003] Recombinant DNA technology comprises extremely varied andpowerful molecular biology techniques aimed at modifying nucleic acidsat the DNA level and makes it possible to analyze and modify genomes atthe molecular level. In this respect, viruses, because of the small sizeof their genome are particularly amenable to such manipulations.However, recombinant DNA technology is not immediately applicable tononretroviral RNA viruses because these viruses do not encompass a DNAintermediate step in their replication. For such viruses, infectiousclones (for instance as a DNA copy or as in vitro transcribed RNA copyor as derivative of either) have to be developed before recombinant DNAtechnology can be applied to their genome to generate modified virus.Infectious clones can be derived through the construction of full-length(genomic length) cDNA (here used in the broad sense of a DNA copy of RNAand not only in the strict sense of a DNA copy of mRNA) of the virusunder study after which an infectious transcript is synthesized in vivoin cells transfected with the full-length cDNA, but infectioustranscripts can also be obtained by in vitro transcription from in vitroligated partial-length cDNA fragments that comprise the full viralgenome. In all cases, the transcribed RNA carries all the modificationsthat have been introduced to the cDNA and can be used to further passagethe thus modified virus.

[0004] Infectious cDNA clones and infectious in vitro transcripts havebeen generated for a great number of positive strand RNA viruses (for areview see Boyer and Haenni, Virology 198, 415-426) with a genome of upto 12 kb or slightly larger. The viral genomic length of Pestivirusesseems, until now, the longest positive strand viral RNA genome fromwhich infectious clones (Moormann et al., J. Vir. 70:763-770) have beenprepared. Problems associated with genomic length lie not only in thedifficulty of obtaining and maintaining long and stabile cDNA clones inbacteria but also in the infectivity of the initial RNA transcript ofwhich replication in the host cell has to be achieved without the helpof the normally associated viral proteins connected with viralreplication. To achieve successful infection, viral transcripts mustinteract with viral-encoded proteins, most particularly with the viralreplicase and with host cell components such as the translationmachinery; therefore, the structure of viral transcripts has to mimicthat of virion RNA as closely as possible. Additional problems can befound with those positive strand RNA viruses that replicate via amechanism of subgenomic messenger RNAs transcribed from the 3′ side ofthe genome and with those positive strand RNA viruses that generateduring replication defective interfering particles, such as nakedcapsids or empty shell particles, comprising several structural proteinsbut only a part of the genome. The presence of incomplete viral RNAfragments or of, for example, matrix or nucleocapsid proteinsinteracting or interfering with the viral RNA to be transcribed or toreplicative intermediate RNA and disrupting its structure will abolishfull-length RNA strand synthesis, and thus the generation of infectiousvirus comprising genomic length RNA.

[0005] “Lelystad virus” (LV), also called “porcine reproductiverespiratory syndrome virus” (PRRSV, genomic length 15.2 kb), is a memberof the family Arteriviridae, which also comprises equine arteritis virus(EAV, genomic length 12.7 kb), lactate dehydrogenase-elevating virus(LDV, genomic length at least 14.2 kb) and simian hemorrhagic fevervirus (SHFV genomic length approximately 15 kb) (Meulenberg et al.,1993a; Plagemann and Moennig, 1993).

[0006] Recently, the International Committee on the Taxonomy of Virusesdecided to incorporate this family in a new order of viruses, theNidovirales, together with the Coronaviridae (genomic length 28 to 30kb), and Toroviridae (genomic length 26 to 28 kb). Nidoviralesrepresents enveloped RNA viruses that contain a positive-stranded RNAgenome and synthesize a 3′ nested set of subgenomic RNAs duringreplication. The subgenomic RNAs of coronaviruses and arterivirusescontain a leader sequence that is derived from the 5′ end of the viralgenome (Spaan et al., 1988; Plagemann and Moennig, 1993). The subgenomicRNAs of toroviruses lack a leader sequence (Snijder and Horzinek, 1993).Whereas the ORFs 1a and 1b, encoding the RNA dependent RNA polymerase,are expressed from the genomic RNA, the smaller ORFs at the 3′ end ofthe genomes of Nidovirales encoding structural proteins are expressedfrom the subgenomic mRNAs.

[0007] PRRSV (Lelystad virus), or “LV”, was first isolated in 1991 byWensvoort et al. (1991). It was shown to be the causative agent of a newdisease now generally known as a porcine reproductive respiratorysyndrome, (“PRRS”). The main symptoms of the disease are respiratoryproblems in pigs and abortions in sows. Although the major outbreaks,such as observed at first in the US in 1987 and in Europe in 1991, havediminished, this virus still causes economic losses in herds in the US,Europe, and Asia.

[0008] PRRSV preferentially grows in alveolar lung macrophages(Wensvoort et al., 1991). A few cell lines, such as CL2621 and othercell lines cloned from the monkey kidney cell line MA-104 (Benfield etal., 1992; Collins et al., 1992; Kim et al., 1993), are also susceptibleto the virus. Some well known PRRSV strains are known under accessionnumbers CNCM I-1102, I-1140, I-1387, I-1388, ECACC V93070108, or ATCC VR2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402. The genome ofPRRSV was completely or partly sequenced (Conzelmann et al., 1993;Meulenberg et al., 1993a, Murthaugh et al, 1995) and encodes, besidesthe RNA dependent RNA polymerase (ORFs 1a and 1b), six structuralproteins of which four envelope glycoproteins named GP₂ (ORF2), GP₃(ORF3), GP₄ (ORF4) and GP₅ (ORF5), a non-glycosylated membrane protein M(ORF6) and the nucleocapsid protein N (ORF7) (Meulenberg et al. 1995,1996; van Nieuwstadt et al., 1996). Immunological characterization andnucleotide sequencing of European and US strains of PRRSV has identifiedminor antigenic differences within strains of PRRSV located in thestructural viral proteins (Nelson et al., 1993; Wensvoort et al., 1992;Murtaugh et al., 1995).

[0009] Pigs can be infected by PRRSV via the oronasal route. Virus inthe lungs is taken up by lung alveolar macrophages and in these cellsreplication of PRRSV is completed within 9 hours. PRRSV travels from thelungs to the lung lymph nodes within 12 hours and to peripheral lymphnodes, bone marrow and spleen within 3 days. At these sites, only a fewcells stain positive for viral antigen. The virus is present in theblood during at least 21 days and often much longer. After 7 days,antibodies to PRRSV are found in the blood. The combined presence ofvirus and antibody in PRRS infected pigs shows that the virus infectioncan persist for a long time, albeit at a low level, despite the presenceof antibody. During at least 7 weeks, the population of alveolar cellsin the lungs is different from normal SPF lungs.

[0010] PRRSV needs its envelope to infect pigs via the oronasal route.The normal immune response of the pig entails, among other things, theproduction of neutralizing antibodies directed against one or more ofthe envelope proteins. Such antibodies can render the virusnon-infective. However, once in the alveolar macrophage, the virus alsoproduces naked capsids, constructed of RNA encapsidated by the M and/orN protein, sometimes partly containing any one of the glycoproteins. Theintra- and extracellular presence of these incomplete viral particles or(partly) naked capsids can be demonstrated by electron microscopy.Sometimes, naked capsids without a nucleic acid content can be found.The naked capsids are distributed through the body by the bloodstreamand are taken up from the blood by macrophages in spleen, lymph nodesand bone marrow. These naked, but infectious, viral capsids cannot beneutralized by the antibodies generated by the pig thus explaining thepersistence of the viral infection in the presence of antibody. In thisway, the macrophage progeny from infected bone marrow cells spreads thevirus infection to new sites in the body. Because not all bone marrowmacrophage-lineage cells are infected, only a small number ofmacrophages at peripheral sites are infected and produce virus.

[0011] PRRSV capsids, consisting of ORF7 proteins only, can be formed inthe absence of other viral proteins by, for instance, infection ofmacrophages with a chimeric pseudorabies-ORF7 vector virus. The PRVvirus was manipulated to contain ORF7 genetic information of PRRSV.After 18 hours post infection, the cytoplasm of infected cells containslarge numbers of small, empty spherical structures with the size of PRRSvirus nucleocapsids.

BRIEF SUMMARY OF THE INVENTION

[0012] The invention provides an infectious clone derived from a viruswith a genomic length far exceeding the maximum genomic length of thepositive strand RNA viruses from which infectious clones have beenobtained so far. The experimental part hereof describes the generationof an infectious clone based on and derived from PRRSV with a genomiclength of 15.2 kb but such clones can now also be obtained from LDV andSHFV that also have a genomic length of about 15 kb and from EAV,although its genome is slightly smaller, and from viruses with greatergenomic length, such as the Coronaviridae or Toroviridae.

[0013] The invention also provides a method to generate infectiousclones by circumventing the problems encountered in viral RNA strandsynthesis associated with the presence of incomplete viral RNA fragmentsor of, for example, matrix or nucleocapsid proteins interacting orinterfering with the to be transcribed RNA transcript or withreplicative intermediate RNA, disrupting the structure that abolishesfull-length RNA strand synthesis, and thus the generation of infectiousvirus.

[0014] The invention provides a method of generating infectious clonesby transfecting a host cell that is, in essence, not susceptible toinfection with the wild-type virus with a recombinant nucleic acid basedon the genome of the virus followed by rescuing infectious progeny virusfrom the host cell by passaging to or cocultivation with cells that aresusceptible to the virus. Cells that are, in essence, not susceptiblemay, in comparison with the cells that are routinely used for thereplication of the virus under study, be only slightly susceptible or benot susceptible at all to the virus under study, but may be fullysusceptible to other virus strains.

[0015] The invention provides a method to generate infectious clones bytransfecting host cells that are not susceptible to infection with thewild-type virus, thus avoiding the generation of naked capsids orincomplete viral particles comprising RNA fragments and matrix ornucleocapsid proteins that interfere with viral RNA strand synthesis.Infectious virus is rescued from the thus transfected host cells bypassaging to cells that are susceptible to the virus. In theexperimental part, hereof, we describe how, in this way, an infectiousclone of PRRSV is obtained, but the method is also applicable to otherpositive strand RNA viruses.

[0016] The invention also provides the possibility of generating amodified infectious clone via the further application of recombinant DNAtechnology. Such modifications may be single or multiple mutations,substitutions, deletions or insertions or combinations thereof that canbe achieved via any recombinant DNA technology method known in the art.The present invention thus provides modified RNA viruses that can beused to investigate RNA viruses and to prepare vaccines.

[0017] The invention also provides infectious clones, for example,derived from Arteriviridae, such as PRRSV, which can be used as asingle-purpose vaccine against the disease caused by the virus fromwhich the infectious clone is based. For example, the infectious clonebased on PRRSV can now be used to study virulence markers or serologicalmarkers of the PRRSV. Known serological markers of PRRSV are, forexample, located on any of the structural proteins of PRRSV encoded byORF2 to ORF7. They can also be found in the proteins encoded by ORF 1aand 1b.

[0018] Virulence markers are present in the ORF 1a and 1b encoding thenonstructural proteins of PRRSV but can also be found on any of theproteins encoded by ORF2 to ORF7. By modifying the genome of theinfectious clone with respect to those markers, it is possible to obtainPRRSV that is not or is much less virulent than its parent strain,and/or that is modified by deleting or introducing serological markersto enable a serological differentiation between vaccinated and wild-typevirus infected pigs. Such modifications are, for instance, provided bythe PRRSV infectious clones in which the nucleic acid sequence encodingthe ORF7 N protein is replaced by the ORF7 protein of ATCC VR2332 orLDV.

[0019] The invention also provides infectious clones, for example,derived from Arteriviridae, such as PRRSV, which can be used as adelivery system or viral vector vaccine for a wide variety of antigens.In such clones, heterologous nucleic acid sequences that do notcorrespond to the sequence of the virus under study are inserted. Suchheterologous nucleic acid sequences can be, for example, derived fromsequences encoding any antigen of choice. The antigen is a protein orpeptide that can induce immunity against a pathogen. Since the virusinfects macrophages and macrophage-lineage cells in bone marrow, anddistributes the antigen-containing virus through its progeny cells, thisviral vector vaccine infects cells central to the immune system and canpresent the antigens for further processing. The vector vaccine virusinfects antigen presenting cells like the dendritic macrophages or theKuppfer cells or other cells of the immune system, and can do this as an(incompletely) enveloped viral particle or as a naked capsid particle.

[0020] Since an infection with a naked capsid or an incomplete virusparticle ensures a persistent infection, the immunological boostereffect will cause a lifelong (because of continuous stimulation on a lowlevel) immunity against pathogens from which the antigens are selected.The virus can be used as an antigen carrier by including in theinformation for epitopes of other pathogenic organisms or substances.Several of such vector vaccine viruses carrying foreign epitopicinformation may be mixed and administered at one time. This enablesactive immunity against several different antigens of one pathogen, oractive immunity against several different pathogens.

[0021] The invention also provides infectious clones, for example,derived from Arteriviridae, such as PRRSV, which can be used as a dualpurpose vaccine. For example, the infectious clone based on PRRSV can beused to construct a vaccine which protects against PRRSV and againstanother pathogen simply by combining the vector vaccine development withthe development directed towards the development of a single purposevaccine directed against PRRS. A specific dual purpose vaccine could bedeveloped that protects against respiratory disease in pigs by insertingin the PRRS vaccine antigens derived from any of the wide variety ofother respiratory pathogens that are known to infect pigs.

[0022] The invention also provides vaccines, be it single purpose, dualpurpose, or vector vaccines, which are relatively safe in the sense thatthe vaccines cannot be shed to the environment. Safety of the vaccines(non-shedding) can be ensured by deleting the information of those viralproteins that is needed to produce enveloped, infectious virus. Thisvirus is propagated in a cell-line that constitutively expresses theprotein. Virus replicating in this complementary cell-line has acomplete envelope, and is capable of infecting pig macrophages. Afterone replication-cycle, the progeny virus, missing the information forthe envelope protein, is no longer capable of infecting other cells asan enveloped virus. Infection of macrophages in the body is stillpossible, as naked capsid or incomplete viral particle.

[0023] The invention also provides viral antigens and proteins that canbe harvested from cell cultures infected with the modified RNA virusesaccording to the invention. Such antigens can be used in diagnosticassays such as ELISA's or other types of diagnostic assay known to theexpert. Such assays can be used as stand-alone tests for primarydiagnosis or as accompanying tests to be applied in animal populationsthat have been vaccinated with a discriminating or marker vaccine basedon the modified RNA viruses according to the invention.

[0024] The invention also provides a PRRSV genome, for example, LDV,VR-2332, or P129 (SEQ ID NO:1), or a sequence that hybridizes to thecomplement under appropriate conditions.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIG. 1. Construction of a genome-length cDNA clone of LV. Theupper part (A) shows the fusion of cDNA clones, which were previouslysequenced (Meulenberg et al., 1993a) in pGEM-4Z. The pABV numbers of theclones and the restriction sites that were used are indicated. The blackboxes represent those parts of the cDNA clones that are fused in thenext cloning step. Light gray boxes, indicated with R.T., are cDNAclones newly generated by RT-PCR; a dark gray box represents a new cDNAclone generated by PCR. The lower part (B) shows the assembly of thelarger cDNA clones pABV331/369, pABV384, and pABV368 with the 5′ endclone pABV396, containing a T7 RNA polymerase promoter, and the 3′ endclone pABV395, containing a poly (A) tail, in low copy number vectorpOK12. The restriction sites within and outside the multiple cloningsite of pOK12 are indicated. The restriction endonuclease sites are; A,ApaI; Ap, ApoI; B, BamHI; Bg, BglII; Bs, BspE1; Bc, BclI; E, EcoRI; Ec,EcoRV; H, HindIII; K, KpnI; N, NarI; Nc, NcoI; S, SacII; Sp, Spel; Sa,SalI; Sc, ScaI; P, PstI; Pm, PmlI; X,XbaI; Xh, XhoI.

[0026]FIG. 2. Terminal sequences of cloned full-length LV cDNA andinfectious RNA transcribed from this cDNA clone. Genome-length cDNAclones were linearized with PvuI and were transcribed in the presence ofthe synthetic cap analog m⁷G (5′) ppp (5′) G with T7 RNA polymerase. Theresulting RNA should contain one extra nucleotide (G) at the 5′ end andtwo extra nucleotides (GC) at the 3′ end. The arrows in the RNAcorrespond to the 5′ and 3′ terminal nucleotides corresponding to theauthentic LV RNA sequence.

[0027]FIG. 3. Growth curves of LV wild-type virus TH, LV4.2.1, andrecombinant viruses vABV414 and vABV416 in porcine alveolar macrophages(A) and CL2621 cells (B). The recombinant viruses vABV414 and vABV416produced in BHK-21 cells were either used directly (BHK), or used aftermultiplication in Porcine alveolar macrophages (PAM). The TH virus wasprepared in porcine alveolar macrophages (PAM), whereas LV4.2.1 wasprepared in CL2621 cells (CL). The cell cultures were infected with theindicated viruses at an MOI of 0.05 and harvested at the indicated timepoints. Virus titers (TCID₅₀/ml) were determined on Porcine alveolarmacrophages or CL2621 cells by end point dilution.

[0028]FIG. 4. Introduction of a unique PacI and SwaI site in theinfectious cDNA clone of LV. The PacI and SwaI sites were created byPCR-directed mutagenesis, as described in detail in Materials andMethods. The cDNA fragments containing the PacI and SwaI site wereexchanged in pABV414 using its unique HpaI and XbaI sites, which areindicated. This resulted in pABV437 and pABV442, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0029] The production of cDNA clones from which infectious RNA can betranscribed in vitro has become an essential tool for molecular geneticanalysis of positive-strand RNA viruses. This technology is applicableto positive-strand RNA viruses whose RNA genomes may function as mRNAand initiate a complete infectious cycle upon introduction intoappropriate host cells. For a number of viruses, infectious clones havebeen described that facilitate studies on the genetic expression,replication, function of viral proteins and recombination of RNA viruses(for a review, see, Boyer and Haenni, 1994). In addition, these clonescan be considered for the development of new viral vectors and vaccines.An infectious cDNA clone has not been described for Arteriviruses sofar. We report here the generation of an infectious clone of PRRSV andits first application in the generation of chimeric PRRS viruses.

[0030] The invention provides an isolated or recombinant nucleic acidcomprising a DNA sequence encoding an infectious RNA molecule encoding agenetically modified PRRS virus. In a exemplary embodiment, the PRRSvirus is genetically modified such that when it infects a porcine animalit is: a) unable to produce PRRS in the animal, and b) able to elicit aneffective immunoprotective response against infection by a PRRSV in theanimal. For example, the DNA sequence may be SEQ ID NO:24, or a sequencehomologous thereto, containing one or more mutations that geneticallydisable the ability of the encoded PRRSV to produce fully functionalviruses. In another exemplary embodiment, the isolated or recombinantnucleic acid may comprise a plasmid or other vector sequence known inthe art.

[0031] Furthermore, when reference is made herein to sequenceshomologous to a sequence, such as SEQ ID NO:24 (a North Americanstrain), it is to be understood that sequences, DNA or RNA, homologousto the sequence in the Sequence Listing and sequences homologous to asequence complementary to the sequence in the Sequence Listing are alsoincluded.

[0032] Homologous sequences, polypeptide or nucleic acid, can bedetermined by comparison of sequences, for example by using BLAST.Alternatively, homologous nucleotide sequences can be determined byhybridization under selected conditions. For example, the nucleotidesequence of a second nucleic acid is homologous to SEQ ID NO:24 if ithybridizes to the complement of SEQ ID NO:24 under conditions which willotherwise result in hybridization of sequences that encode a PRRSV. Inan exemplary embodiment, a second nucleotide sequence is homologous toSEQ ID NO:24 if it hybridizes to the complement of SEQ ID NO:24 underconditions, for example, hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C. (see, Ausubel et al., 1989, Current Protocols In MolecularBiology, Greene Publishing Associates & Wiley Interscience, NY)(Ausubelet al.).

[0033] Cells and Viruses

[0034] The Ter Huurne strain of PRRSV (or LV) (deposited at CNCM, Paris,under accession number I-1102) was isolated in 1991 (Wensvoort et al.,1991) and was grown in primary alveolar macrophages or in CL2621 cells.Passage 6 of the Ter Huurne strain (TH) was used in this study as wellas a derivative of this strain, LV4.2. 1, which was adapted for growthon CL2621 cells by serial passage. Alveolar macrophages were maintainedin RPMI 1640 growth medium (Flow), whereas CL2621 cells were maintainedin Hank's minimal essential medium (Gibco-BRL/Life technologies). BHK-21cells were maintained in Dulbecco's minimal essential medium. Fortransfection experiments, BHK-21 cells were grown in Glasgow minimalessential medium (GIBCO-BRL/Life Technologies Ltd), according to themethod of Liljeström and Garoff (1993).

[0035] Isolation of Viral RNAs

[0036] Intracellular RNA was isolated from alveolar macrophages orCL2621 cells 24 hours after infection with PRRSV at a multiplicity ofinfection of 1, as described earlier (Meulenberg et al., 1993a). Inorder to isolate virion genomic RNA, virions were purified on sucrosegradients as described by van Nieuwstadt et al. (1996) and wereresuspended in TNE (0.01 M Tris-HCl, pH 7.2, 0.1 M NaCl, 1 mM EDTA). Oneml of Proteinase Kbuffer (100 mM Tris-HCl, pH 7.2, 25 mM EDTA, 300 mMNaCl, 2% (w/v) SDS) and 0.4 mg Proteinase K (Boehringer Mannheim) wasadded to one ml of purified PRRSV virions (10⁸ TCID₅₀). This reactionmixture was incubated at 37° C. for 30 min. The RNA was extracted oncewith phenol/chloroform (1:1) and precipitated with ethanol. The RNA wasstored in ethanol at −20° C. One tenth of this RNA preparation was usedin Reversed Transcription (RT) reactions.

[0037] Cloning of the 5′ and 3′ Termini of the PRRSV Genome.

[0038] The 5′ end of the viral genome of PRRSV was cloned using amodified single strand ligation to single-stranded cDNA procedure (SLIC;Edwards et al., 1991). One tenth of the virion RNA, prepared asdescribed above, was used in a RT reaction with primer 11U113 (5′TACAGGTGCCTGATCCAAGA 3′) (SEQ ID NO: 1) that is complementary tonucleotides 1232 to 1251 of the genome. The RT reaction was performed ina final volume of 20 ml, as described earlier (Meulenberg et al.,1993b). Subsequently, 2 ml 6M NaOH was added to the RT-reaction and theRNA was hydrolyzed for 30 min at 37° C. The single strand cDNA waspurified using the high pure PCR Product Purification Kit of BoehringerMannheim. The purified cDNA was precipitated with ethanol, resuspendedin TE, and ligated to an anchor primer ALG3(5′CACGAATTCACTATCGATTCTGGATCCTTC 3′) (SEQ ID NO: 2). This primercontains an EcoRI, ClaI, and BamHI site, and its 3′ end is modified withan amino blocking group to prevent self-ligation. The single strand cDNAproduct was ligated to 4 pmol ALG3 in 50 mM Tris-HCl (pH 8.0), 10 mMMgCl₂, 10 mg/ml BSA, 25% PEG, 1.0 mM Hexamine Cobalt chloride, 40 mMATP, and 0.5 ml (10 U) T4 RNA ligase (New England Biolabs), overnight atroom temperature. One third of the ligation reaction was used astemplate in a PCR with primers LV69 (5′ AGGTCGTCGACGGGCCCCGTGATCGGGTACC3′) (SEQ ID NO: 3) and ALG4 (5′ GAAGGATCCAGAATCGATAG 3′) (SEQ ID NO: 4).Primer LV69 is complementary to nucleotides 594 to 615 of the LV genome,whereas ALG4 is complementary to anchor primer ALG3. The PCR conditionswere as described in Meulenberg et al. (1993b) and the obtained productwas digested with EcoRI and SalI and cloned in pGEM-4Z. A similarstrategy was used to clone the 5′ terminus of the LV genome fromintracellular LV RNA. For these experiments 10 mg of total cellular RNAisolated from CL2621 cells infected with LV was used. The 5′ cDNA cloneswere sequenced and one clone, pABV387, containing an extension of 10nucleotides compared to the published PRRSV sequence (Meulenberg et al.,1993a), was used for further experiments.

[0039] A 3′ end cDNA clone containing a long poly (A) tail wasconstructed by reverse transcription of LV RNA with primer LV76 (5′TCTAGGAATTCTAGACGATCG(T)₄₀ 3′) (SEQ ID NO: 5), which contains an EcoRI,XbaI, and PvuI site. The reversed transcription reaction was followed bya PCR with primers LV75 (5′ TCTAGGAATTCTAGACGATCGT 3′ ) (SEQ ID NO: 6),which is identical to LV76 except for the poly(T) stretch, and 39U70R(5′ GGAGTGGTTAACCTCGTCAA 3′) (SEQ ID NO: 7), a sense primercorresponding to nucleotides 14566-14585 of the LV genome and containingan HpaI site. The resulting PCR products were digested with HpaI andEcoRI and cloned in cDNA clone pABV39 restricted with the same enzymes(FIG. 1). Two cDNA clones containing a poly(A) stretch of 45 A's(pABV382) and 109 A's (pABV392) and the correct genomic cDNA sequence,as assessed by oligonucleotide sequencing, were used to construct thefull length genomic cDNA clone.

[0040] Sequence Analysis.

[0041] Oligonucleotide sequences were determined with the PRISM™ ReadyReaction Dye Deoxy™ Terminator Cycle Sequencing Kit and Automaticsequencer of Applied Biosystems.

[0042] Construction of Full-Length Genomic cDNA Clones of PRRSV.

[0043] cDNA clones generated earlier to determine the nucleotidesequence of the genome of LV (Meulenberg et al., 1993a), were ligatedtogether at convenient restriction sites as shown in FIG. 1. PlasmidpABV254 was constructed from pABV clones 25, 11, 12, and 100 and wasused in a previous study (den Boon et al., 1996). Standard cloningprocedures were carried out according to Sambrook et al. (1989). Thisresulted in three plasmids containing overlapping cDNA sequences of LVin high copy number plasmid pGEM-4Z. Plasmids pABV331 and pABV369consist of nucleotides 5 to 6015 of the LV genome. A nucleotidedifference was found at position 3462 at a ratio of 1:1 in a set of 6independent cDNA clones that were sequenced in that region. Thisnucleotide difference resulted in an amino acid substitution at position1084 in ORF1A (Leu instead of Pro). Since we could not predict theinfluence of this amino acid on infectivity, we also cloned the Leuencoding cDNA fragment in pABV331 by exchange at the EcoRV (nucleotide3403) and SacII (nucleotide 3605) site, which resulted in pABV369.Plasmid pABV384 consists of nucleotides 5168 to 9825 of the LV genome.Since no appropriate cDNA clone was yet available that had overlap withplasmids pABV20 and pABV5, and could finally be fused to the cDNAsequences of pABV331 and pABV369, two new cDNA fragments were generatedby RT-PCR. Sense primer LV59 (5′ TCGGAATCTAGATCTCACGTGGTGCAGCTGCTG 3′)(SEQ ID NO: 8) corresponding to nucleotides 5169-5186 and antisenseprimer 61U303 (5′ CATCAACACCTGTGCAGACC 3′) (SEQ ID NO: 9) complementaryto nucleotides 6078 to 6097 were used in one PCR. Sense primer 61U526R(5′ TTCCTTCTCTGGCGCATGAT 3′) (SEQ ID NO: 10) located at nucleotides 5936to 5955 and LV60 (5′ GTACTGGTACCGGATCCGTGAGGATGTTGC 340 ) (SEQ ID NO:11) complementary to nucleotides 6727 to 6745 were used in another PCR.These two PCR fragments were ligated together in pABV20 using the XbaIsite incorporated in LV59, the internal ApoI site (nucleotides 6006) andthe BamHI site at nucleotide 6740, which was also incorporated in primerLV60. The new cDNA fragment was completely sequenced and did not containany mutations that resulted in amino acid differences with the publishedsequence (Meulenberg et al, 1993a). Plasmid pABV368 encompassesnucleotides 8274 to 13720 of the PRRSV genome. Since further ligation ofcDNA fragments in pGEM-4Z resulted in instable clones, the inserts ofpABV331/369, pABV384, and pABV368 were ligated to the 5′ and 3′ cDNAfragments in pOK12 (Viera and Messing, 1991). Plasmid vector pOK12 isexpected to be more suitable for cloning of large foreign cDNAsequences, because it has a lower copy number than pGEM-4Z. Plasmidswere transformed to Escherichia coli strain DH5a, grown at 32° C. in thepresence of 15 mg/ml Kanamycin, to keep the copy number as low aspossible. First, the cDNA fragments of pABV382 ((A)₄₅) and pABV392((A)₁₀₉) were excised by digestion with EcoRI and modification of thissite with Klenow polymerase (Pharmacia) to a blunt end, followed bydigestion with BamHI. These fragments were cloned in pOK12 digested withBamHI and FspI, the latter site also modified to a blunt end, resultingin pABV394 and pABV395. In this way, the T7 RNA polymerase promoterpresent in pOK12 was removed. Subsequently, the cDNA fragments ofpABV368 and pABV384 were ligated to the 3′ end cDNA clones using theBclI site (nucleotide 13394), the ScaI site (nucleotide 8657) and theBamHI and BglII sites in flanking or vector sequences. This resulted inplasmids pABV401 and pABV402 (FIG. 1).

[0044] A 5′ cDNA clone, containing the T7 RNA polymerase promoterdirectly fused to the 5′ terminus of the LV genome, was amplified by PCRfrom pABV387 with primers LV83 (5′GAATTCACTAGTTAATACGACTCACTATAGATGATGTGTAGGGTATTCC 3′ ) (SEQ ID NO: 12)and LV69. LV83 is composed of, in order from 5′ to 3′, an EcoRI and SpeIsite, a T7 RNA polymerase promoter sequence, a single G for initiationof transcription, and nucleotides 1 to 19 of the LV genome. The PCRfragment was cloned in the EcoRI and SalI site of pOK12, resulting inpABV396. The correct sequence of pABV396 was assessed by oligonucleotidesequencing. Subsequently, the LV cDNA fragments of pABV331 and pABV369were excised with ApaI and BamHI, and were ligated to pABV396, digestedwith ApaI and BamHI. Finally, the resulting 5′ cDNA fragments werecloned into pABV401 and pABV402, using the Spel site upstream of the T7RNA polymerase promoter and the unique PmlI site at position 5168 in theviral genome. In this way, genome-length cDNA clones were obtained ascorresponding to viruses resembling the parent strain and to chimericviruses comprising foreign open reading frames.

[0045] Production of Mutant Viruses Containing a PacI and/or SwaI Site

[0046] To introduce a unique PacI site in the genome-length cDNA clonedirectly downstream of the ORF7 gene, the T and A at nucleotides 14987and 14988 were both replaced by an A in a PCR using sense primer LV108(5′ GGAGTGGTTAACCTCGTCAAGTATGGCCGGTAAAAACCAGAGCC3′) (SEQ ID NO: 13) withantisense primer LV 112 (5′CCATTCACCTGACTGTTTAATTAACTTGCACCCTGA3′) (SEQID NO: 14) and sense primer LV111(5′TCAGGGTGCAAGTTAATTAAACAGTCAGGTGAATGG 3′) (SEQ ID NO: 15) with LV75.Similarly, a unique SwaI site was created by changing the G at position14980 for a T, and the T at position 14985 for an A by PCR with primersLV108 and LV110 (5′CCTGACTGTCAATTTAAATTGCACCCTGAC 3′) (SEQ ID NO: 16)and primers LV109 (5′GTCAGGGTGCAATTTAAATTGACAGTCAGG 3′) (SEQ ID NO: 17)and LV111. The PCR fragments were ligated in pABV395 using the createdPacI and SwaI site and flanking HpaI and XbaI sites, resulting inpABV427 and pABV426, respectively. This fragment was then inserted inpABV414 using the same unique HpaI and XbaI sites, resulting in pABV437and pABV442 (see, FIG. 4). To detect the marker mutation in the virusrecovered from transcripts of pABV437 and pABV422, RNA was isolated fromthe supernatant of infected porcine alveolar macrophages. This RNA wasused in reverse transcription-PCR to amplify a fragment approximately0.6 kb (spanning nucleotides 14576-polyA tail of variable length) withprimers LV76, LV75 and 39U70R. The presence of the genetic marker wasdetected by digesting the PCR fragments with PacI or SwaI.

[0047] In vitro Transcription and Transfection of RNA

[0048] Plasmids pABV414, pABV416, containing the full-length genomiccDNA fragment of LV, were linearized with PvuI, which is locateddirectly downstream of the poly(A) stretch. Plasmid pABV296, whichconsists of ORF4 in Semliki Forest virus (SFV) expression vector pSFVI(Meulenberg et al., 1997), was linearized with SpeI and served ascontrol for in vitro transcription and transfection experiments. Thelinearized plasmids were precipitated with ethanol and 1.5 mg of theseplasmids was used for in vitro transcription with T7 RNA polymerase(plasmids pABV414, pABV416) or Sp6 RNA polymerase (pABV296), accordingto the methods described for SFV by Liljeström and Garoff (1991 and1993). The in vitro transcribed RNA was precipitated with isopropanol,washed with 70% ethanol and stored at −20° C. until use. BHK-21 cellswere seeded in M6 wells (approximately 10⁶ cells/well) and transfectedwith 2.5 mg RNA mixed with 10 ml lipofectin in optimem as described byLiljeström and Garoff (1993). Alternatively, RNA was introduced inBHK-21 cells by electroporation. In this case, 10 Cg in vitrotranscribed RNA or 10 Cg intracellular LV RNA was transfected toapproximately 10⁷ BHK-21 cells using the electroporation conditions ofLiljeström and Garoff (16). The medium was harvested 24 hours aftertransfection and transferred to CL2621 cells to rescue infectious virus.Transfected and infected cells were tested for expression of LV-specificproteins by an immunoperoxidase monolayer assay (IPMA), essentially asdescribed by Wensvoort et al. (1986). Monoclonal antibodies (MAbs)122.13, 122.59, 122.9 and 122.17, directed against the GP₃, GP₄, M and Nprotein (van Nieuwstadt et al., 1996) were used for staining in theIPMA.

[0049] Reconstruction of the 5′ Terminal Sequence of the Genomic RNA ofLV.

[0050] Although the infectivity of in vitro-transcribed RNAs withtruncated 5′ ends have been reported (Davis et al. 1989, Klump et al.,1990), it is generally admitted that the entire viral sequence,including the utmost 5′ and 3′ end, are required to obtain infectiousclones. To clone the 5′ end of the LV genome, a modified single strandligation to single-stranded cDNA (SLIC; Edwards et al., 1991) procedurewas used. Both intracellular RNA isolated from CL2621 cells infectedwith LV and LV RNA from purified virions was reverse transcribed usingprimer LV69, which was complementary to the 5′ end of ORF1A. The firststrand cDNA product was ligated to an anchor primer ALG3 of which the 3′end was blocked for self ligation. The ligated products were amplifiedby PCR and cloned. Twelve clones, derived from LV intracellular RNA andresulting from two independent PCRs, and fourteen clones derived fromvirion RNA and resulting from two independent PCRs were sequenced. Fromthese 26 cDNA clones, 22 clones contained an extension of 10 nucleotides(5′ ATGATGTGTA 3′) (SEQ ID NO: 18) compared to the cDNA sequence,published previously (Meulenberg et al., 1993a), whereas four cloneslacked one to three nucleotides at the 5′ end of this additionalsequence (Table 1). This led us to conclude that these ten nucleotidesrepresent the utmost 5′ end of the LV genome and was thereforeincorporated in the genome-length cDNA clone.

[0051] Construction of Genome-Length cDNA Clones of LV

[0052] In order to construct a genome-length cDNA clone of LV, cDNAsthat were isolated and sequenced previously (Meulenberg et al., 1993a)were joined at shared restriction enzyme sites, according to thestrategy depicted in FIG. 1. In addition, new cDNA fragments weregenerated to assemble the genome-length cDNA clones. One cDNA fragmentspanning nucleotides 5168 to 6740 was created by RT-PCR to enable theligation of cDNA sequences from clones pABV5 and pABV20. A T7 RNApolymerase promoter for in vitro transcription was directly linked tothe 5′ terminus of the genome of LV by PCR and this new cDNA fragment,cloned in pABV396, and was inserted in the genome-length cDNA clone.Resequencing of nucleotides 3420 to 3725 on six new and independent cDNAclones indicated that at amino acid 1084 in ORF1a a Leu and Pro arepresent at a ratio of 1:1. Since we could not predict the influence ofthis amino acid on the infectivity of the RNA transcribed from the finalgenome-length cDNA clone, we used both to construct this clone. At the3′ end, two different cDNA clones were used. We had previously isolated3′ end cDNA clones containing poly(A) tails of at maximum 20 A's(Meulenberg et al., 1993a). However, in view of studies reported on thelength of poly(A) tails of related viruses such as LDV (Chen et al.,1994), the entire poly(A) tail was expected to be much longer.Therefore, new 3′ end cDNA clones were generated using primer LV76 thatcontains a stretch of 40 T residues. These cDNA clones were sequencedand contained stretches of 40 to 109 A residues. The cDNA clonecontaining the longest poly(A) stretch (109 A residues; pABV392) wasused for the genome-length cDNA clone. Since long homo-polymeric tractsmight interfere with the replication of plasmids in E. coli (Deng andWu, 1981), we also selected a second clone, pABV382, containing 45 Aresidues for use in subsequent cloning steps. Previously, it wasobserved that maintenance of genome-length cDNA clones in high copynumber plasmids leads to accumulation of mutations or deletions thatresults in loss of infectivity of transcripts synthesized from theseclones (Lai et al., 1991; Rice et al., 1987; Sumiyoshi et al., 1992). Wealso observed instability of plasmids, when we tried to ligate thelarger cDNA fragments of pABV clones 331/369,384, and 368 to the 5′ and3′ end in pGEM-4Z and, therefore, we finally fused these clones to eachother in low copy number vector pOK12 (Viera and Messing, 1991). Thisresulted in the genome-length cDNA clones pABV414/415 and 416, whichcould be stably propagated in E. coli under the growth conditions used.No difference in stability of the genome-length cDNA clones containing45 or 109 A residues was observed.

[0053] Infectivity of LV RNA

[0054] LV, preferentially, grows in porcine alveolar macrophages. Thusfar, cell line CL2621 or other clones derived from the monkey kidneycell line MA104, are cell lines which have been shown to propagate LV(Benfield et al., 1992; Collins et al., 1992; Kim et al., 1993).Therefore, CL2621 cells were used to determine the optimal conditionsfor transfection of LV RNA.

[0055] RNA isolated from CL2621 cells infected with LV was transfectedto CL2621 cells at different doses using different methods, such aslipofectin, lipofectamin, DEAE-dextran and electroporation. Cells werescreened for cythopathic effect and plaques until 7 days posttransfection, but these signs of infectious virus could not be detected.In addition, no LV-specific antigens could be detected in IPMA usingLV-specific MAbs. RNA transcribed in vitro from pABV296 was used ascontrol in these experiments. Plasmid pABV296 consists of the ORF4 geneencoding GP₄ inserted in expression vector pSFVI (Meulenberg et al.,1997).

[0056] The transfection efficiency of the pABV296 RNA was tested bystaining of the transfected cells in IPMA with GP₄-specific MAbs. Thehighest transfection efficiency, resulting in 0.01% positive CL2621cells, was obtained by electroporation, whereas 80-90% positive cellswere obtained using similar conditions with BHK-21 cells.

[0057] These results indicated that CL2621 cells were not suitable fortransfection experiments, whereas the BHK-21 cells (not susceptible toinfection with wild-type virus) surprisingly appeared very suitable.Therefore BHK-21 cells were used to test the infectivity of LV RNA. Twomg of RNA isolated from CL2621 cells infected with LV was transfected toapproximately 10⁶ BHK-21 cells with lipofectin, according to theconditions described for SFV (Liljeström and Garoff, 1993).

[0058] Twenty-four hours after transfection, cells were stained withLV-specific MAb 122.17 directed against the N protein of LV.Approximately 3-10 individual cells were stained positive, but noinfectious centers or plaques suggesting cell to cell spread wereobserved. Transfection of the control RNA transcribed from pABV296resulted in 60-70% positive BHK-21 cells using these conditions. Thesupernatant of the BHK-21 cells transfected with intracellular LV RNAand pABV296 RNA were transferred to CL2621 cells.

[0059] After 3 to 4 days, plaques were observed in the cells that wereincubated with the supernatant from BHK-21 cells transfected withintracellular LV RNA, but not in those incubated with supernatant fromBHK-21 cells transfected with pABV296 RNA. The plaques were positivelystained with LV-specific MAbs in IPMA. Similar results were obtainedwhen RNA isolated from purified virions of LV was used. Furthermore, thenumber of positively stained cells increased 2 to 4 fold when cells weretransfected by electroporation.

[0060] These data indicated that LV can not infect BHK-21 cells because,most likely, they lack the receptor for LV. However, once the genomicRNA has been introduced in BHK-21 cells, new infectious virus particlesare being produced and excreted into the medium. Reinfection of alreadytransfected BHK-21 cells with these particles being naked capsids orfully or partly enveloped particles is again not possible.

[0061] In vitro Synthesis of Infectious RNA.

[0062] Since the BHK-21 cells, which are in essence not susceptible toinfection by wild-type PRRSV, were specifically appropriate for therescue of virus from intracellular LV RNA and the susceptible CL2621cells were not, BHK-21 cells were used to test whether RNA transcribedfrom the genome-length cDNA clones was infectious. Plasmids pABV414/416were linearized with PvuI and transcribed in vitro using T7 RNApolymerase. The PvuI site is located directly downstream of the poly(A)stretch, such that the transcribed RNA contains 2 non-viral nucleotidesat the 3′ end (FIG. 2). In addition, transcripts should contain anon-viral G at the 5′ end, which is the transcription start site of T7RNA polymerase. Approximately 2.5 mg of in vitro transcribed RNA wastransfected to BHK-21 cells, together with 2 mg intracellular LV RNA asa positive control for subsequent virus rescue in CL2621 cells, andpABV296 RNA as a positive control for RNA transfection to BHK-21 cellsand negative control for subsequent virus rescue in CL2621 cells. At 24hours after transfection, the supernatant of the cells was harvested andthe cells were fixed and stained in IPMA with N-specific MAb 122.17.Whereas only a few positive cells were observed in the wells with BHK-21cells that were transfected with intracellular LV RNA, 800 to 2700positive cells were observed in the wells with BHK-21 cells transfectedwith RNA transcribed from pABV414/416. In order to check whetherinfectious virus was released from the cells, the supernatants were usedto infect CL2621 cells. Plaques were produced in CL2621 cultures thatwere infected with the supernatant from BHK-21 cells transfected withintracellular LV RNA and transcripts of pABV414/415. The plaques stainedpositive in IPMA with MAbs against the N, M, GP₄, and GP₃ protein,suggesting that these proteins were all properly expressed. No plaquesand staining in IPMA were observed in CL2621 cultures incubated with thesupernatant of BHK-21 cells transfected with RNA transcribed frompABV296. Therefore, these results clearly show that transfection of RNAtranscribed from genome-length cDNA clones pABV414 and pABV416 to BHK-21cells results in the production and release of infectious LV. Moreover,when transcripts of pABV414 and pABV416 were transfected to BHK-21 cellsby electroporation instead of lipofectin, a two- to four fold increaseof cells staining positive with LV-specific MAbs was obtained. The titerof the recombinant viruses in the supernatant of these electroporatedBHK-21 cells was approximately 10⁵ TCID₅₀/ml.

[0063] Growth Curves of Infectious Copy Virus Compared to Ter Huurne andLV4.2.1 Growth characteristics of Rescued Virus

[0064] The initial transfection and infection experiments suggested thatthe rescued recombinant viruses, designated vABV414 and vABV416, infectand grow equally well in porcine alveolar macrophages, but grow sloweron CL2621 cells than the virus rescued from BHK-21 cells transfectedwith intracellular LV RNA. This intracellular LV RNA was isolated fromCL2621 cells infected with LV4.2. 1, which has been adapted for growthon CL2621. To study the growth properties of vABV414 and vABV416 morethoroughly, growth curves were determined in CL2621 cells and porcinealveolar macrophages and were compared with those of wild-type LV thathas only been passaged on porcine alveolar macrophages (TH) and withthose of LV4.2.1 grown on CL2621 cells. The growth rates of the tworecombinant viruses did not differ, growing equally well regardless ofwhether they were derived directly from BHK-21 or further passaged onporcine alveolar macrophages (FIG. 3). Titers (7.1-7.9 TCID₅₀/ml) inporcine alveolar macrophages peaked around 32 hours post infection,whereas the titers in CL2621 where slower and had not yet peaked even at96 hours post infection. TH virus had growth characteristics similar tothe recombinants. In contrast, the CL2621-adapted virus LV4.2.1 grewfaster on CL2621 cells than the viruses vABV414, vABV416 and TH (FIG.3).In summary, these results demonstrate that the growth properties of therecombinant viruses are similar to those of the TH virus. This wasexpected, since the cDNA sequence used to construct the infectiousclones was derived from the parental “non-adapted” TH virus.

[0065] Introduction of a Genetic Marker in the Infectious Clone of LV

[0066] To demonstrate that the genome-length cDNA clone can be used togenerate mutant LV viruses, a unique PacI and SwaI site was introduceddirectly downstream of the ORF7 gene by PCR-directed mutagenesis (FIG.4). When RNA transcribed from the genome-length cDNA clone pABV437containing the PacI site and pABV442 containing the SwaI site wastransfected to BHK-21 cells and the supernatant was transferred toporcine alveolar macrophages and CL2621 cells at 24 hours aftertransfection, infectious virus was produced. The rescued viruses,vABV437 and vABV442, had similar growth properties in porcine alveolarmacrophages and CL2621 cells as the parental virus vABV414 (data notshown). A specific region of approximately 0.6 kb (nucleotides14576-poly(A) tail) was amplified by reverse transcription and PCR ofviral RNA isolated from the supernatant of porcine alveolar macrophagesinfected with vABV414 and vABV416. Digestion with PacI showed that thisrestriction site was indeed present in the fragment derived from vABV437but was absent from the fragment derived from vABV414. Similarly, thepresence of SwaI site in vABV442 was demonstrated (data not shown).Thus, we were able to exclude the possibility of contamination withwild-type virus and therefore we confirmed the identity of vABV437 andvABV442.

Best Mode

[0067] Modern recombinant DNA technology allows us to analyze and modifygenomes at the molecular level and thus gain deeper insight into theirorganization and expression. In the case of RNA viruses, this requiresthe generation of genome-length cDNA clones from which infectioustranscripts can be synthesized. In most instances, a prerequisite forthe construction of infectious clones is the identification of thesequences at the termini of the respective viral genome that areprobably crucial for replication of viral RNA. In a previous study, itwas shown that LV contains a poly(A)tail at the 3′ end (Meulenberg etal, 1993a). In the present work, the exact 5′ end of the LV genome wasdetermined. Whereas several methods have been described to determine the5′ end of viral genomic RNAs or mRNAs, but most of them have importantlimitations. For flaviruses and pestiviruses, a method has been usedwhich is based on the circularization of genomic RNA. However, thismethod needs accompanying analyses to define the border between the 5′and 3′ end of the genome. The 5′ rapid amplification of cDNA ends (5′RACE) method is based on the addition of a homopolymeric tail withterminal deoxyribonucleotide transferase (TdT) to the first strand cDNAstrand. However, the tailing reaction is rather inefficient and thismethod also requires additional analyses since it can not be concludedwhether the first nucleotide of the tail represents the viral sequenceor is already part of the enzymatically added tail. As described above,we have determined the utmost 5′ end of the viral genome by ligation ofan oligonucleotide with a specified sequence to a first strand primerextension product and amplification by PCR. An extension of 10nucleotides (ATGATGTGTA) (SEQ ID NO: 18) with respect to the publishedsequence was found in several independent clones and was thereforeassumed to represent the utmost 5′ end nucleotides of the viral genome.Altogether, this results in a leader sequence of 221 nucleotides, whichis similar in length to the leader of EAV (207 nucleotides; den Boon etal., 1991), SHFV (208 nucleotides; Zeng et al., 1995), but longer thanthe leader of LDV (155 nucleotides; Chen et al., 1994). However, nosignificant homology exists between the leader sequences of thesearteriviruses.

[0068] The utmost 5′ end was incorporated in genome-length cDNA tocreate an infectious clone. Major problems with the generation ofinfectious clones concern the stability of the virus sequences whencloned in bacteria as well as the generation of the correct 5′ and 3′termini. Although initial attempts to assemble a genome-length cDNAclone in pGEM-4Z failed, the methods and principles of the presentinvention produced the 15,207 nucleotides long genomic cDNA fragment ofLV which remained stable in low copy number plasmid pOK12. As notedabove this cDNA fragment is now the longest infectious clone of apositive RNA strand virus thus far generated. Transcripts of thegenomic-length cDNA clones contained a 5′ cap structure and an extranon-viral G at the 5′ end and a nonviral CG at the 3′ end, but theseextensions did not abolish their infectivity. Several investigators havereported a reduced initial infection of RNA transcribed from full-lengthcDNA clones due to extraneous, non-authentic sequences at either the 5′or 3′ ends or to incomplete capping. Transcripts of LV full-length cDNAlacking a cap structure were not infectious. Whereas the infectivity oftranscripts of infectious cDNA clones have always been tested in celllines that are susceptible to the virus, we were unable to demonstratethe infectivity of transcripts from genome-length cDNA clones or LV RNAisolated from CL2621 cells by transfection of these RNAs to CL2621cells. This was due to the poor transfection efficiency in CL2621 cells,whereby viral RNA strand synthesis is probably hampered by interferenceor interaction with incomplete RNA fragments or capsid proteinsresulting from reinfection of the CL2621 cells with defectiveinterfering particles such as naked capsids containing only fragments ofthe viral genome. However, transfection of transcripts from full-lengthcDNA clones and intracellular LV RNA to BHK-21 resulted in theproduction and release of infectious virus that could be rescued inCL2621 cells. Reinfection of BHK-21 cells with naked capsids does notoccur and thus does not hamper full-length viral RNA synthesis. Thespecific infectivity was roughly 400-1500 positive cells per mg in vitrotranscribed RNA, whereas 2 to 5 positive cells were obtained per mg LVintracellular RNA. However, these specific infectivities can not becompared because only a very small fraction of the intracellular RNAisolated from LV-infected CL2621 cells represent genomic LV RNA.Furthermore, the amount of genomic RNA isolated from virions that wasused for transfections was too small to allow accurate quantification.

[0069] In addition, BHK-21 cells were scored for antigen production inIPMA with LV-specific MAbs, which does not necessarily correlate withproduction of infectious virus. This was clear from the fact that thesupernatant of BHK-21 cells transfected with 2 mg intracellular LV RNAcontained a higher titer of plaque forming units assayed on CL2621 cellsthan the supernatant of BHK-21 cells transfected with 2.5 mg transcriptof full-length cDNA clones. Although it was shown previously for anumber of viruses that the length of the poly(A) tail influenced theinfectivity of the viral transcripts (Holy and Abouhaidar, 1993; Sarow,1989), we did not observe any difference in infectivity betweentranscripts from genomic cDNA clones containing a tail of 45 or 109residues. It might be possible that a tail of 45 A residues is above athreshold length below which stability of the corresponding transcriptswill be altered. We have found a clone difference at amino acid 1084 inORF1a, giving a PRO and LEU at a ratio of 1:1. This amino acid did nothave an influence on infectivity since transcripts of full-length cDNAclones containing this LEU or PRO codon did not display any differencein infectivity of BHK-21 cells.

[0070] The genome-length infectious clone was used to generate achimeric virus expressing the nucleocapsid protein of PRRSV strain ATCCVR2332. In addition, the genome-length infectious clone was used togenerate a chimeric virus expressing the nucleocapsid protein of themouse virus LDV. The chimeric viruses can be distinguished from parentalviruses with strain-specific MAbs. They do not stain with monoclonalantibodies specifically reactive with the N (ORF7) protein of the TerHuurne strain of PRRSV. Furthermore, the chimeric virus in which thePRRSV N protein is substituted with the LDV N protein is not reactivewith porcine convalescent antibodies reactive with the PRRSV N protein.Since all PRRSV infected pigs develop antibodies directed against thePRRSV N protein, the chimeric viruses can be used for future projectsusing new live vaccines against PRRSV, making use of this virus as avector system which is specifically targeted to its host cell, thealveolar lung macrophage. In this respect, it should be mentioned thatinitial attempts to confer protection with killed virus or recombinantsubunits were disappointing. The up-to-date, only effective, vaccineagainst PRRS available is a modified live vaccine based on a US strain(Gorcyca, et al., 1995). However, pigs vaccinated with this modifiedlive product can not be discriminated from pigs infected with fieldvirus. The infectious clone of PRRSV thus provides a so-called markervaccine by site-directed mutagenesis of the genome, such that vaccinatedpigs can be distinguished from field virus-infected pigs on the basis ofdifference in serum antibodies. A distinguishing assay can thus befashioned using methods known to those skilled in the art.

[0071] The infectious clone of LV, described here, is the longestinfectious clone ever developed of a positive strand RNA virus and thefirst of the arterivirus family. The generation of this infectious cloneof PRRSV opens up new opportunities for studies directed at thepathogenesis, host tropism, and replication and transcription of thisvirus. Arteriviruses and coronaviruses share a specific transcriptionmechanism also referred to as leader primed transcription which involvesthe generation of a so-called nested set of subgenomic RNAs containing acommon 5′ leader (Spaan et. al., 1988; Plagemann and Moennig, 1991).This leader primed transcription is a complex process that is not yetfully understood. Studies of coronavirus virologist to elucidate theunderlying mechanism of leader-primed transcription are restricted toanalyses and site directed mutagenesis of cDNAs of defecting interferingRNAs, since the large size of the genome (28 to 30 kb) has impeded theconstruction of an infectious clone. The infectious clone of PRRSV thusprovides a model system to study and unravel the intriguing mechanism oftranscription and replication of arteriviruses and coronaviruses.

[0072] Infectious clones derived from PRRSV can also be used as adelivery system or vector vaccine virus for foreign antigens inserted inthe PRRSV genome because the virus infects macrophages andmacrophage-lineage cells in bone marrow and other cells of the immunesystem and distribute the antigen-containing virus through its progenycells. In the specific instance of antigens containing fragments of theORF7 or N protein of Arteriviruses or PRRSV, these antigens will be(over)expressed at the outer side of the cell membrane of the infectedcell, thereby further enhancing the immune response. Such immunologicalbooster effects will cause a lifelong (because of continuous stimulationon a low level) immunity against pathogens. We can use the virus as anantigen carrier by building in the information for epitopes of otherpathogenic organisms or substances. Several modified PRRS virusescarrying foreign epitopic information may be mixed and administered atone time. This enables active immunity against several differentepitopes of one pathogen, or active immunity against several differentpathogens. Safety of the modified PRRSV vaccines (such as non-shedding)can be ensured by deleting the information of those viral proteins thatare needed to produce enveloped, infectious virus. This virus has to bepropagated in a cell-line that constitutively expresses that envelopeprotein. Virus replicating in this complementary cell-line has acomplete envelope and is capable of infecting macrophages in the pig.After one replication-cycle, the progeny virus, missing the informationfor the envelope protein, is no longer capable of infecting other cellsas a fully enveloped virus. Infection of macrophages in the body isstill possible as naked capsid. In this way, the vaccine will becontained to the animal that has been vaccinated and will not spread toother animals.

References

[0073] Benfield, D. A., E. Nelson, E. Collins, J. E., Harris, L., Goyal,S. M., Robison, D., Christianson, W. T., Morrison, R. B., Gorcyca, D.E., and Chladek, D. W. (1992). Characterization of swine infertility andrespiratory syndrome virus (Isolate ATCC-VR2332) J. Vet. Diagn. Invest.4, 127-133.

[0074] Boyer, J., and Haenni, A. (1994) Infectious transcripts and cDNAclones of RNA viruses. Virology, 198, 415-426.

[0075] Chen, Z., Faaberg, K. S., and Plagemann, P. G. W. (1994)Determination of the 5′ end of the lactate dehydrogenase-elevating virusgenome by two independent approaches. J. Gen. Virol. 75, 925-930.

[0076] Collins, J. E., Benfield, D. A., Christianson, W. T., Harris, L.,Hennings, J. C., Shaw, D. P., Goyal, S. M., McCullough, S., Morrison, R.B., Joo, H. S., Gorcyca, D. E., Chladek, D. W. (1992). Isolation ofswine infertility and respiratory syndrome virus (Isolate ATCC-VR-2332)in North America and experimental reproduction of the disease ingnotobiotic pigs. J. of Vet. Diagn. Invest. 4, 117-126.

[0077] Conzelmann, K. K., Visser, N., van Woensel, P., and Tiel, H. J.(1993). Molecular characterization of porcine reproductive andrespiratory syndrome virus, a member of the Arterivirus group. Virology193, 329-339.

[0078] den Boon, J. A., Faaberg, K. S., Meulenberg, J. J. M., Wassenaar,A. L. M., Plagemann, P. G. W., Gorbalenya, A. E., and Snijder, E. J.(1995) Processing and evolution of the N-terminal region of thearterivirus replicase ORF1a protein: identification of two papainlikecysteine proteases. J. Virol. 69: 4500-4505.

[0079] Davis, N. L., Willis, L. V., Smith, J. F., and Johnston, R. E.(1989). In vitro synthesis of infectious Venezuelan equine encephalitisvirus RNA of an insect virus. Proc. Natl. Acad. Sci. USA 83, 63-66.

[0080] Deng, R., and Wu, R. (1981). An improved procedure for utilizingterminal transferase to add homopolymers to the 3′ termini of DNA.Nucleic Acids Res. 9, 4173-4188.

[0081] Edwards, J. B. D. M., Delort, J., and Mallet, J. (1991)Oligodeoxyribonucleotide ligation to single-stranded cDNAs; A new toolfor cloning 5′ ends of mRNAs and for constructing cDNA libraries by invitro amplification. Nucleic Acids Res. 19, 5227-5232.

[0082] Gorcyca, D., Schlesinger, K., Chladek, D., et al., (1995) Proc.Am. Assoc. of Swine Pract., Omaha, Nebr., 1-22.

[0083] Holy, S., and Abouhaidar, M. G. (1993). Production of infectiousin vitro transcripts from a full-length clover yellow mosaic virus cDNAclone. J. Gen. Virol., 74, 781-784.

[0084] Kim, H. S., Kwang, J., and Yoon, I. Y. (1993). Enhancedreplication of porcine reproductive and respiratory syndrome virus in ahomogeneous subpopulation of MA-104 cell line. Arch. Virol. 133,477-483.

[0085] Klump, W. M., Bergmann, I., Muller, B. C., Ameis, D., andKandolf, R. (1990) Complete nucleotide sequence of infectiouscoxsackie-virus B3 cDNA: Two initial 5′uridine residues are regainedduring plus-strand RNA synthesis. J. Virol. 64, 1573-1583.

[0086] Lai, C. J., Zhao, B., Hori, H., and Bray, M. (1991) InfectiousRNA transcribed from stably cloned full-length cDNA of dengue type 4virus. Proc. Natl. Acad. Sci. USA 88, 5139-5143.

[0087] Liljeström, P. and Garoff, H. (1991). A new generation of animalcell expression vectors based on the Semliki Forest virus replicon.Biotechnol. 9, 1356-1361.

[0088] Liljeström, P., and Garoff, H. (1993) Expression of proteinsusing Semliki Forest virus vectors, p. 16.xx.1-16.xx.00 In: Currentprotocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston,D. D. Moore, J. A. Smith, J. G. Seidman and K. Struhl (Eds.). GreenePublishing associates and Wiley Interscience, New York.

[0089] Meulenberg, J. J. M., Hulst, M. M., de Meijer, E. J., Moonen, P.L. J. M., den Besten, A., de Kluyver, E. P., Wensvoort, G., andMoormann, R. J. M. (1993a). Lelystad virus, the causative agent ofporcine epidemic abortion and respiratory syndrome (PEARS) is related toLDV and EAV. Virology 192, 62-74.

[0090] Meulenberg, J. J. M., de Meijer, E. J., and Moorrnann, R. J. M.(1993b). Subgenomic RNAs of Lelystad virus contains a conserved junctionsequence. J. of Gen. Virol. 74, 1697-1701.

[0091] Meulenberg, J. J. M., Petersen-den Besten, A., de Kluyver, E. P.,Moormann, R. J. M., Wensvoort, G (1995). Characterization of proteinsencoded by ORFs 2 to 7 of Lelystad virus. Virology 206, 155-163.

[0092] Meulenberg, J. J. M., and Petersen-den Besten (1996)Identification and characterization of a sixth structural protein ofLelystad virus: The glycoprotein GP₂ encoded by ORF2 is incorporated invirus particles. Virology, in press.

[0093] Meulenberg et al., 1997.

[0094] Murtaugh, M. P., Elam, M. R., and Kakach (1995) Comparison of thestructural protein coding sequences of the VR-2332 and Lelystad virusstrains of the PRRS virus. Arch. Virol., 140, 1451-1460.

[0095] Nelson, E. A., Christopher-Hennings, J., Drew, T., Wensvoort, G.,Collins, J. E., and Benfield, D. A. (1993). Differentiation of Unitedstates and European isolates of porcine reproductive and respiratorysyndrome virus by monoclonal antibodies. J. of Clin. Microbiol. 31,3184-3189.

[0096] Plagemann, P. G. W., and Moennig, V. (1991). Lactatedehydrogenase-elevating virus, equine arteritis virus, and simianhemorrhagic fever virus: a new group of positive-strand RNA viruses.Adv. in Virus Res. 41, 99-192.

[0097] Rice, C. M., Levis, R., Strauss, J. H., and Huang, H. V. (1987).Production of infectiuos RNA transcripts from Sindbis virus cDNA clones:mapping of lethal mutations, rescue of a temperature-sensitive marker,and in vitro mutagenesis to generate defined mutants. J. Virol., 61,3809-3819.

[0098] Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). MolecularCloning, A Laboratory Manual. Cold Spring Harbor Lab., Cold SpringHarbor, N.Y.

[0099] Sarnow, P. (1989) Role of 3′ end sequences in infectivity ofpolio-virus transcripts made in vitro. J. Virol., 63, 467-470.

[0100] Snijder, E. J., and Horzinek, M. C. (1993). Toroviruses:replication, evolution and comparison with other members of thecoronavirus-like superfamily. J. Gen. Virol., 74, 2305-2316.

[0101] Spaan, W. J. M., Cavanagh, D., and Horzinek, M. C. (1988)Coronaviruses: Structure and genome expression. J. Gen. Virol. 69,2939-2952.

[0102] Sumiyoshi, H., Hoke, C. H., and Trent, D. W. (1992). InfectiousJapanese encephalitis virus RNA can be synthesized from in vitro-ligatedcDNA templates. J. Virol., 66, 5425-5431.

[0103] van Nieuwstadt, A. P., Meulenberg, J. J. M., vanEssen-Zandbergen, A., Petersen-den Besten, A., Bende, R. J., Moormann,R. J. M., and Wensvoort, G. (1996). Proteins encoded by ORFs 3 and 4 ofthe genome of Lelystad virus (Arteriviridae) are structural proteins ofthe virion. J. Virol., 70, 4767-4772.

[0104] Viera, J., and Messing, J. (1991) New pUC-derived cloning vectorswith different selectable markers and DNA replication origins. Gene,100, 189-194.

[0105] Wensvoort, G., de Kluyver, E. P., Luijtze, E. A., de Besten, A.,Harris, L., Collins, J. E., Christianson, W. T., and Chladek, D. (1992)Antigenic comparison of Lelystad virus and swine infertility andrespiratory (SIRS) virus. J. Vet. Diagn. Invest. 4, 134-138.

[0106] Wensvoort, G., Terpstra, C., Boonstra, J., Bloemraad, M., and VanZaane, D. (1986) Production of monoclonal antibodies against swine fevervirus and their use in laboratory diagnosis. Vet. Microbiol. 12,101-108.

[0107] Wensvoort, G., Terpstra, C., Pol, J. M. A., Ter Laak, E. A.,Bloemraad, M., de Kluyver, E. P., Kragten, C., van Buiten, L., denBesten, A., Wagenaar, F., Broekhuijsen, J. M., Moonen, P. L. J. M.,Zetstra, T., de Boer, E. A., Tibben, H. J., de Jong, M. F., van't Veld,P., Groenland, G. J. R., van Gennep, J. A., Voets, M. Th., Verheijden,J. H. M., and Braamskamp, J. (1991). Mystery swine disease in theNetherlands: the isolation of Lelystad virus. Vet. Quart. 13, 121-130.

[0108] Zeng, L., Godeny, E. K., Methven, S. L., and Brinton, M. A.(1995) Analysis of simian hemorrhagic fever virus (SHFV) subgenomicRNAs, junction sequences and 5′ leader. Virology 207, 543-548. TABLE 1Nucleotide sequence of 5′ end clones of LV. Sequence¹ No. of clonesATGATGTGTAGGG..... 22  TGATGTGTAGGG.....  1   GATGTGTAGGG.....  2   ATGTGTAGGG.....  1

[0109]

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 32 <210> SEQ ID NO 1<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer 11U113 <400> SEQUENCE: 1tacaggtgcc tgatccaaga 20 <210> SEQ ID NO 2 <211> LENGTH: 30 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Anchor primer ALG3 <400> SEQUENCE: 2 cacgaattca ctatcgattc tggatccttc 30<210> SEQ ID NO 3 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: Primer LV69 <400>SEQUENCE: 3 aggtcgtcga cgggccccgt gatcgggtac c 31 <210> SEQ ID NO 4<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer ALG4 <400> SEQUENCE: 4gaaggatcca gaatcgatag 20 <210> SEQ ID NO 5 <211> LENGTH: 22 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Primer LV76 <400> SEQUENCE: 5 tctaggaatt ctagacgatc gt 22 <210> SEQ IDNO 6 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer LV75 <400> SEQUENCE: 6tctaggaatt ctagacgatc gt 22 <210> SEQ ID NO 7 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: Sense primer 39U7OR <400> SEQUENCE: 7 ggagtggtta acctcgtcaa20 <210> SEQ ID NO 8 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: Sense primer LV59<400> SEQUENCE: 8 tcggaatcta gatctcacgt ggtgcagctg ctg 33 <210> SEQ IDNO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Antisense primer 61U303 <400>SEQUENCE: 9 catcaacacc tgtgcagacc 20 <210> SEQ ID NO 10 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: Sense primer 61U526R <400> SEQUENCE: 10 ttccttctctggcgcatgat 20 <210> SEQ ID NO 11 <211> LENGTH: 30 <212> TYPE: DNA <213>ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION: Primer LV60<400> SEQUENCE: 11 gtactggtac cggatccgtg aggatgttgc 30 <210> SEQ ID NO12 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Primer LV83 <400> SEQUENCE: 12gaattcacta gttaatacga ctcactatag atgatgtgta gggtattcc 49 <210> SEQ ID NO13 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Sense primer LV108 <400> SEQUENCE: 13ggagtggtta acctcgtcaa gtatggccgg taaaaaccag agcc 44 <210> SEQ ID NO 14<211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: Antisense primer LV112 <400> SEQUENCE:14 ccattcacct gactgtttaa ttaacttgca ccctga 36 <210> SEQ ID NO 15 <211>LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: Sense primer LV111 <400> SEQUENCE: 15tcagggtgca agttaattaa acagtcaggt gaatgg 36 <210> SEQ ID NO 16 <211>LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: Primer LV110 <400> SEQUENCE: 16 cctgactgtcaatttaaatt gcaccctgac 30 <210> SEQ ID NO 17 <211> LENGTH: 30 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Primer LV109 <400> SEQUENCE: 17 gtcagggtgc aatttaaatt gacagtcagg 30<210> SEQ ID NO 18 <211> LENGTH: 10 <212> TYPE: DNA <213> ORGANISM:Artificial <220> FEATURE: <223> OTHER INFORMATION: 5′ prime end of thegenome <400> SEQUENCE: 18 atgatgtgta 10 <210> SEQ ID NO 19 <211> LENGTH:37 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHERINFORMATION: 5′ end 1 <400> SEQUENCE: 19 taatacgact cactatagatgatgtgtagg gtattcc 37 <210> SEQ ID NO 20 <211> LENGTH: 111 <212> TYPE:DNA <213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:3′ end <400> SEQUENCE: 20 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaac g 111 <210> SEQ ID NO 21 <211> LENGTH: 6 <212> TYPE: DNA<213> ORGANISM: Artificial <220> FEATURE: <223> OTHER INFORMATION:Reverse 3′ end <400> SEQUENCE: 21 cgatcg 6 <210> SEQ ID NO 22 <211>LENGTH: 121 <212> TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:<223> OTHER INFORMATION: 3′end <400> SEQUENCE: 22 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaac gatcgtctag 120 a 121 <210> SEQ ID NO 23<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial <220>FEATURE: <223> OTHER INFORMATION: 5′ end <400> SEQUENCE: 23 augauguguaggguauucc 19 <210> SEQ ID NO 24 <211> LENGTH: 15450 <212> TYPE: DNA<213> ORGANISM: Arterivirus porcine respiratory and reproductivesyndrome virus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(192)..(7685) <223> OTHER INFORMATION: ORF 1a <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (12057)..(12827) <223> OTHER INFORMATION:GP2 (ORF 2) <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(13225)..(13761) <223> OTHER INFORMATION: GP4 (ORF 4) <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (13772)..(14374) <223> OTHERINFORMATION: GP5 (ORF 5) <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (14873)..(15244) <223> OTHER INFORMATION: protein N (ORF 7)<400> SEQUENCE: 24 atgacgtata ggtgttggct ctatgccacg gcatttgtattgtcaggagc tgtgaccatt 60 ggcacagccc aaaacttgct gcacggaaaa cgcccttctgtgacagcctt cttcagggga 120 gcttaggggt ctgtccctag caccttgctt ctggagttgcactgctttac ggtctctcca 180 cccctttaac c atg tct ggg ata ctt gat cgg tgcacg tgc acc ccc aat 230 Met Ser Gly Ile Leu Asp Arg Cys Thr Cys Thr ProAsn 1 5 10 gcc agg gtg ttt atg gcg gag ggc caa gtc tac tgc aca cga tgtctc 278 Ala Arg Val Phe Met Ala Glu Gly Gln Val Tyr Cys Thr Arg Cys Leu15 20 25 agt gca cgg tct ctc ctt cct ctg aat ctc caa gtt cct gag ctt ggg326 Ser Ala Arg Ser Leu Leu Pro Leu Asn Leu Gln Val Pro Glu Leu Gly 3035 40 45 gtg ctg ggc cta ttt tat agg ccc gaa gag cca ctc cgg tgg acg ttg374 Val Leu Gly Leu Phe Tyr Arg Pro Glu Glu Pro Leu Arg Trp Thr Leu 5055 60 cca cgt gca ttc ccc act gtc gag tgc tcc ccc gcc ggg gcc tgc tgg422 Pro Arg Ala Phe Pro Thr Val Glu Cys Ser Pro Ala Gly Ala Cys Trp 6570 75 ctt tct gcg atc ttt cca att gca cga atg acc agt gga aac ctg aac470 Leu Ser Ala Ile Phe Pro Ile Ala Arg Met Thr Ser Gly Asn Leu Asn 8085 90 ttt caa caa aga atg gtg cgg gtt gca gct gag att tac aga gcc ggc518 Phe Gln Gln Arg Met Val Arg Val Ala Ala Glu Ile Tyr Arg Ala Gly 95100 105 caa ctc acc cct gca gtt ttg aag gct cta caa gtt tat gaa cgg ggt566 Gln Leu Thr Pro Ala Val Leu Lys Ala Leu Gln Val Tyr Glu Arg Gly 110115 120 125 tgt cgc tgg tac ccc att gtc gga cct gtc cct gga gtg gcc gttcac 614 Cys Arg Trp Tyr Pro Ile Val Gly Pro Val Pro Gly Val Ala Val His130 135 140 gcc aac tcc cta cat gtg agt gac aaa cct ttc ccg gga gca actcat 662 Ala Asn Ser Leu His Val Ser Asp Lys Pro Phe Pro Gly Ala Thr His145 150 155 gtg tta acc aac cta ccg ctc ccg cag agg ccc aag cct gaa gacttt 710 Val Leu Thr Asn Leu Pro Leu Pro Gln Arg Pro Lys Pro Glu Asp Phe160 165 170 tgc cct ttt gag tgt gct atg gct gac gtc tat gac att agc catgac 758 Cys Pro Phe Glu Cys Ala Met Ala Asp Val Tyr Asp Ile Ser His Asp175 180 185 gcc gtc atg tat gtg gcc aga ggg aaa gtc tcc tgg gcc cct cgtggc 806 Ala Val Met Tyr Val Ala Arg Gly Lys Val Ser Trp Ala Pro Arg Gly190 195 200 205 ggg gat gaa gtg aaa ttt gaa acc gtc ccc gaa gag ttg aagttg att 854 Gly Asp Glu Val Lys Phe Glu Thr Val Pro Glu Glu Leu Lys LeuIle 210 215 220 gcg aac cga ctc cac atc tcc ttc ccg ccc cac cac gca gtggac atg 902 Ala Asn Arg Leu His Ile Ser Phe Pro Pro His His Ala Val AspMet 225 230 235 tct gag ttt gcc ttc ata gcc cct ggg agt ggt gtc tcc ttgcgg gtc 950 Ser Glu Phe Ala Phe Ile Ala Pro Gly Ser Gly Val Ser Leu ArgVal 240 245 250 gag cac caa cac ggt tgc ctt ccc gct gat act gtc cct gaaggg aac 998 Glu His Gln His Gly Cys Leu Pro Ala Asp Thr Val Pro Glu GlyAsn 255 260 265 tgc tgg tgg tgc ttg ttt gac ttg ctc cca ccg gaa gtt cagaat aaa 1046 Cys Trp Trp Cys Leu Phe Asp Leu Leu Pro Pro Glu Val Gln AsnLys 270 275 280 285 gaa att cgc cgt gct aac caa ttt ggc tat caa acc aagcat ggt gtc 1094 Glu Ile Arg Arg Ala Asn Gln Phe Gly Tyr Gln Thr Lys HisGly Val 290 295 300 cct ggc aag tac cta cag cgg agg ctg caa gtt aat ggtctc cga gca 1142 Pro Gly Lys Tyr Leu Gln Arg Arg Leu Gln Val Asn Gly LeuArg Ala 305 310 315 gtg act gat aca gat gga cct att gtc gta cag tac ttctct gtt agg 1190 Val Thr Asp Thr Asp Gly Pro Ile Val Val Gln Tyr Phe SerVal Arg 320 325 330 gag agt tgg atc cgc cac ttc aga ctg gcg gaa gaa cctagc ctc cct 1238 Glu Ser Trp Ile Arg His Phe Arg Leu Ala Glu Glu Pro SerLeu Pro 335 340 345 ggg ttt gaa gac ctc ctc aga ata agg gta gag cct aatacg tcg cca 1286 Gly Phe Glu Asp Leu Leu Arg Ile Arg Val Glu Pro Asn ThrSer Pro 350 355 360 365 ttg ggt ggc aag ggt gaa aaa atc ttc cgg ttt ggcagt cac aag tgg 1334 Leu Gly Gly Lys Gly Glu Lys Ile Phe Arg Phe Gly SerHis Lys Trp 370 375 380 tac ggt gct gga aag aga gca agg aga gca cgc tctggt gca act gcc 1382 Tyr Gly Ala Gly Lys Arg Ala Arg Arg Ala Arg Ser GlyAla Thr Ala 385 390 395 acg gtc gct cac tgc gct ttg ccc gct cgc gaa gcccag cag gcc aag 1430 Thr Val Ala His Cys Ala Leu Pro Ala Arg Glu Ala GlnGln Ala Lys 400 405 410 aag ctc gag gtt gcc agc gcc aac agg gct gag catctc aag tac tat 1478 Lys Leu Glu Val Ala Ser Ala Asn Arg Ala Glu His LeuLys Tyr Tyr 415 420 425 tcc ccg cct gcc gac ggg aac tgt ggt tgg cac tgcatt tcc gcc att 1526 Ser Pro Pro Ala Asp Gly Asn Cys Gly Trp His Cys IleSer Ala Ile 430 435 440 445 acc aac cgg atg gtg aat tcc aaa ttt gaa accact ctt ccc gag aga 1574 Thr Asn Arg Met Val Asn Ser Lys Phe Glu Thr ThrLeu Pro Glu Arg 450 455 460 gtg aga cct tca gat gac tgg gct act gac gaggat ctt gtg aat acc 1622 Val Arg Pro Ser Asp Asp Trp Ala Thr Asp Glu AspLeu Val Asn Thr 465 470 475 atc caa atc ctc agg ctc ccc gcg gcc ttg gacagg aac ggt gct tgt 1670 Ile Gln Ile Leu Arg Leu Pro Ala Ala Leu Asp ArgAsn Gly Ala Cys 480 485 490 gct ggc gcc aag tac gtg ctc aag ctg gaa ggtgag cac tgg acc gtc 1718 Ala Gly Ala Lys Tyr Val Leu Lys Leu Glu Gly GluHis Trp Thr Val 495 500 505 tct gtg acc cct ggg atg acc cct tct ttg ctcccc ctt gaa tgt gtt 1766 Ser Val Thr Pro Gly Met Thr Pro Ser Leu Leu ProLeu Glu Cys Val 510 515 520 525 cag ggt tgt tgt gag cat aag agc ggt cttggt ttc cca gac gtg gtc 1814 Gln Gly Cys Cys Glu His Lys Ser Gly Leu GlyPhe Pro Asp Val Val 530 535 540 gaa gtt tcc gga ttt gac cct gcc tgt cttgac cga ctt gct gag ata 1862 Glu Val Ser Gly Phe Asp Pro Ala Cys Leu AspArg Leu Ala Glu Ile 545 550 555 atg cac tta cct agc agt gtc atc cca gctgct ctg gcc gag atg tcc 1910 Met His Leu Pro Ser Ser Val Ile Pro Ala AlaLeu Ala Glu Met Ser 560 565 570 gac gac ttc aat cgt ctg gct tcc ccg gccgcc act gtg tgg act gtt 1958 Asp Asp Phe Asn Arg Leu Ala Ser Pro Ala AlaThr Val Trp Thr Val 575 580 585 tcg caa ttc ttt gcc cgc cac aga gga ggagag cat cct gac cag gtg 2006 Ser Gln Phe Phe Ala Arg His Arg Gly Gly GluHis Pro Asp Gln Val 590 595 600 605 tgc tta ggg aaa att atc aac ctt tgtcag gtg att gag gaa tgc tgc 2054 Cys Leu Gly Lys Ile Ile Asn Leu Cys GlnVal Ile Glu Glu Cys Cys 610 615 620 tgt tcc cgg aac aaa gcc aac cgg gctacc ccg gaa gag gtt gcg gca 2102 Cys Ser Arg Asn Lys Ala Asn Arg Ala ThrPro Glu Glu Val Ala Ala 625 630 635 aaa gtt gac cag tac ctc cgt ggt gcagca agc ctt gga gaa tgc ttg 2150 Lys Val Asp Gln Tyr Leu Arg Gly Ala AlaSer Leu Gly Glu Cys Leu 640 645 650 gcc aag ctt gag agg gct cgc ccg ccgagc gcg atg gac acc tcc ttt 2198 Ala Lys Leu Glu Arg Ala Arg Pro Pro SerAla Met Asp Thr Ser Phe 655 660 665 gat tgg aat gtt gtg ctt cct ggg gttgag acg gcg gat cag aca acc 2246 Asp Trp Asn Val Val Leu Pro Gly Val GluThr Ala Asp Gln Thr Thr 670 675 680 685 aaa cag ctc cat gtc aac cag tgccgc gct ctg gtt cct gtc gtg act 2294 Lys Gln Leu His Val Asn Gln Cys ArgAla Leu Val Pro Val Val Thr 690 695 700 caa gag cct ttg gac aga gac tcggtc cct ctg acc gcc ttc tcg ctg 2342 Gln Glu Pro Leu Asp Arg Asp Ser ValPro Leu Thr Ala Phe Ser Leu 705 710 715 tcc aat tgc tac tac cct gca caaggt gac gag gtc cgt cac cgt gag 2390 Ser Asn Cys Tyr Tyr Pro Ala Gln GlyAsp Glu Val Arg His Arg Glu 720 725 730 agg cta aac tcc gtg ctc tct aagttg gag ggg gtt gtt cgt gag gaa 2438 Arg Leu Asn Ser Val Leu Ser Lys LeuGlu Gly Val Val Arg Glu Glu 735 740 745 tat ggg ctc acg cca act gga cctggc ccg cga ccc gca ctg ccg aac 2486 Tyr Gly Leu Thr Pro Thr Gly Pro GlyPro Arg Pro Ala Leu Pro Asn 750 755 760 765 ggg ctc gac gag ctt aaa gaccag atg gag gag gat ctg ctg aaa tta 2534 Gly Leu Asp Glu Leu Lys Asp GlnMet Glu Glu Asp Leu Leu Lys Leu 770 775 780 gtc aac gcc cag gca act tcagaa atg atg gcc tgg gca gcc gag cag 2582 Val Asn Ala Gln Ala Thr Ser GluMet Met Ala Trp Ala Ala Glu Gln 785 790 795 gtt gat cta aaa gct tgg gtcaaa aat tac cca cgg tgg aca ccg cca 2630 Val Asp Leu Lys Ala Trp Val LysAsn Tyr Pro Arg Trp Thr Pro Pro 800 805 810 ccc cct cca cca aga gtt cagcct cga aaa acg aag tct gtc aag agc 2678 Pro Pro Pro Pro Arg Val Gln ProArg Lys Thr Lys Ser Val Lys Ser 815 820 825 ttg cta gag aac aag cct gtccct gct ccg cgc agg aag gtc aga tct 2726 Leu Leu Glu Asn Lys Pro Val ProAla Pro Arg Arg Lys Val Arg Ser 830 835 840 845 gat tat ggc agc ccg attttg atg ggc gac aat gtt cct aac ggt tgg 2774 Asp Tyr Gly Ser Pro Ile LeuMet Gly Asp Asn Val Pro Asn Gly Trp 850 855 860 gaa gat tcg act gtt ggtggt ccc ctt gac ctt tcg gca cca tcc gag 2822 Glu Asp Ser Thr Val Gly GlyPro Leu Asp Leu Ser Ala Pro Ser Glu 865 870 875 ccg atg aca cct ctg agtgag cct gta ctt att tcc agg cca gtg aca 2870 Pro Met Thr Pro Leu Ser GluPro Val Leu Ile Ser Arg Pro Val Thr 880 885 890 tct ttg agt gtg ccg gcccca gtt cct gca ccg cgt aga gct gtg tct 2918 Ser Leu Ser Val Pro Ala ProVal Pro Ala Pro Arg Arg Ala Val Ser 895 900 905 cga ccg atg acg ccc tcgagt gag cca att ttt gtg tct gca ctg cga 2966 Arg Pro Met Thr Pro Ser SerGlu Pro Ile Phe Val Ser Ala Leu Arg 910 915 920 925 cac aaa ttt cag caggtg gaa aaa gca aat ctg gcg gca gca gcg ccg 3014 His Lys Phe Gln Gln ValGlu Lys Ala Asn Leu Ala Ala Ala Ala Pro 930 935 940 atg tac cag gac gaaccc tta gat ttg tct gca tcc tca cag act gaa 3062 Met Tyr Gln Asp Glu ProLeu Asp Leu Ser Ala Ser Ser Gln Thr Glu 945 950 955 tat ggg gct tct ccccta aca cca ccg cag aac gtg ggc att ctg gag 3110 Tyr Gly Ala Ser Pro LeuThr Pro Pro Gln Asn Val Gly Ile Leu Glu 960 965 970 gta agg ggg caa gaagct gag gaa gtt ctg agt gaa atc tcg gat att 3158 Val Arg Gly Gln Glu AlaGlu Glu Val Leu Ser Glu Ile Ser Asp Ile 975 980 985 ctg aat gat acc aaccct gca cct gtg tca tca agc agc tcc ctg tca 3206 Leu Asn Asp Thr Asn ProAla Pro Val Ser Ser Ser Ser Ser Leu Ser 990 995 1000 1005 agt gtt aggatc aca cgc cca aaa tac tca gct caa gcc att atc 3251 Ser Val Arg Ile ThrArg Pro Lys Tyr Ser Ala Gln Ala Ile Ile 1010 1015 1020 gac ttg ggc gggccc tgc agt ggg cac ctc caa agg gaa aaa gaa 3296 Asp Leu Gly Gly Pro CysSer Gly His Leu Gln Arg Glu Lys Glu 1025 1030 1035 gca tgc ctc cgc atcatg cgt gag gct tgt gat gcg gcc aag ctt 3341 Ala Cys Leu Arg Ile Met ArgGlu Ala Cys Asp Ala Ala Lys Leu 1040 1045 1050 agt gac cct gcc acg caggaa tgg ctt tct cgc atg tgg gat agg 3386 Ser Asp Pro Ala Thr Gln Glu TrpLeu Ser Arg Met Trp Asp Arg 1055 1060 1065 gtg gac atg ctg act tgg cgcaac acg tct gct tac cag gcg ttt 3431 Val Asp Met Leu Thr Trp Arg Asn ThrSer Ala Tyr Gln Ala Phe 1070 1075 1080 cgc acc tta gat ggc agg ttt gggttt ctc cca aag atg ata ctc 3476 Arg Thr Leu Asp Gly Arg Phe Gly Phe LeuPro Lys Met Ile Leu 1085 1090 1095 gag acg ccg ccg ccc tac ccg tgt gggttt gtg atg ttg cct cac 3521 Glu Thr Pro Pro Pro Tyr Pro Cys Gly Phe ValMet Leu Pro His 1100 1105 1110 acc cct gca cct tcc gtg agt gca gag agcgac ctt acc atc ggt 3566 Thr Pro Ala Pro Ser Val Ser Ala Glu Ser Asp LeuThr Ile Gly 1115 1120 1125 tca gtc gcc act gaa gat att cca cgc atc ctcggg aaa ata gaa 3611 Ser Val Ala Thr Glu Asp Ile Pro Arg Ile Leu Gly LysIle Glu 1130 1135 1140 aat acc ggt gag atg atc aac cag gga ccc ttg gcatcc tct gag 3656 Asn Thr Gly Glu Met Ile Asn Gln Gly Pro Leu Ala Ser SerGlu 1145 1150 1155 gaa gaa ccg gta tac aac caa cct gcc aaa gac tcc cggata tcg 3701 Glu Glu Pro Val Tyr Asn Gln Pro Ala Lys Asp Ser Arg Ile Ser1160 1165 1170 tcg cgg ggg tct gac gag agc aca gca gct ccg tcc gca ggtaca 3746 Ser Arg Gly Ser Asp Glu Ser Thr Ala Ala Pro Ser Ala Gly Thr1175 1180 1185 ggt ggc gcc ggc tta ttt act gat ttg cca cct tca gac ggcgta 3791 Gly Gly Ala Gly Leu Phe Thr Asp Leu Pro Pro Ser Asp Gly Val1190 1195 1200 gat gcg gac ggt ggg ggg ccg ttg cag acg gta aga aag aaagct 3836 Asp Ala Asp Gly Gly Gly Pro Leu Gln Thr Val Arg Lys Lys Ala1205 1210 1215 gaa agg ctc ttc gac caa ttg agc cgt cag gtt ttt aac ctcgtc 3881 Glu Arg Leu Phe Asp Gln Leu Ser Arg Gln Val Phe Asn Leu Val1220 1225 1230 tcc cat ctc cct gtt ttc ttc tca cac ctc ttc aaa tct gacagt 3926 Ser His Leu Pro Val Phe Phe Ser His Leu Phe Lys Ser Asp Ser1235 1240 1245 ggt tat tct ccg ggt gat tgg ggt ttt gca gct ttt act ctattt 3971 Gly Tyr Ser Pro Gly Asp Trp Gly Phe Ala Ala Phe Thr Leu Phe1250 1255 1260 tgc ctc ttt ttg tgt tac agc tac cca ttc ttc ggt ttc gttccc 4016 Cys Leu Phe Leu Cys Tyr Ser Tyr Pro Phe Phe Gly Phe Val Pro1265 1270 1275 ctc ttg ggt gta ttt tct ggg tct tct cgg cgt gtg cgc atgggg 4061 Leu Leu Gly Val Phe Ser Gly Ser Ser Arg Arg Val Arg Met Gly1280 1285 1290 gtt ttt ggc tgc tgg ctg gct ttt gct gtt ggc ctg ttc aagcct 4106 Val Phe Gly Cys Trp Leu Ala Phe Ala Val Gly Leu Phe Lys Pro1295 1300 1305 gtg tcc gac cca gtc ggc act gct tgt gag ttt gac tcg ccagag 4151 Val Ser Asp Pro Val Gly Thr Ala Cys Glu Phe Asp Ser Pro Glu1310 1315 1320 tgt agg aac gtc ctt cat tct ttt gag ctt ctc aaa cct tgggac 4196 Cys Arg Asn Val Leu His Ser Phe Glu Leu Leu Lys Pro Trp Asp1325 1330 1335 cct gtt cgc agc ctt gtt gtg ggc ccc gtc ggt ctc ggt cttgcc 4241 Pro Val Arg Ser Leu Val Val Gly Pro Val Gly Leu Gly Leu Ala1340 1345 1350 att ctt ggc agg tta ctg ggc ggg gca cgc tac atc tgg catttt 4286 Ile Leu Gly Arg Leu Leu Gly Gly Ala Arg Tyr Ile Trp His Phe1355 1360 1365 ttg ctt agg ctt ggc att gtt gca gat tgt atc ttg gct ggagct 4331 Leu Leu Arg Leu Gly Ile Val Ala Asp Cys Ile Leu Ala Gly Ala1370 1375 1380 tat gtg ctt tct caa ggt agg tgt aaa aag tgc tgg gga tcttgt 4376 Tyr Val Leu Ser Gln Gly Arg Cys Lys Lys Cys Trp Gly Ser Cys1385 1390 1395 ata aga act gct cct aat gaa atc gcc ttc aac gtg ttc cctttt 4421 Ile Arg Thr Ala Pro Asn Glu Ile Ala Phe Asn Val Phe Pro Phe1400 1405 1410 aca cgt gcg acc agg tcg tca ctc atc gac ctg tgc gat cggttt 4466 Thr Arg Ala Thr Arg Ser Ser Leu Ile Asp Leu Cys Asp Arg Phe1415 1420 1425 tgt gcg cca aaa ggc atg gac ccc att ttc ctc gcc act gggtgg 4511 Cys Ala Pro Lys Gly Met Asp Pro Ile Phe Leu Ala Thr Gly Trp1430 1435 1440 cgt ggg tgc tgg acc ggc cga agt ccc att gag caa ccc tctgaa 4556 Arg Gly Cys Trp Thr Gly Arg Ser Pro Ile Glu Gln Pro Ser Glu1445 1450 1455 aaa ccc atc gcg ttc gcc cag ttg gat gaa aag agg att acggct 4601 Lys Pro Ile Ala Phe Ala Gln Leu Asp Glu Lys Arg Ile Thr Ala1460 1465 1470 aga act gtg gtc gct cag cct tat gat cct aat caa gcc gtaaag 4646 Arg Thr Val Val Ala Gln Pro Tyr Asp Pro Asn Gln Ala Val Lys1475 1480 1485 tgc ttg cgg gtg tta cag gcg ggt ggg gcg atg gtg gcc gaggca 4691 Cys Leu Arg Val Leu Gln Ala Gly Gly Ala Met Val Ala Glu Ala1490 1495 1500 gtc cca aaa gtg gtc aaa gtt tct gct att cca ttc cga gccccc 4736 Val Pro Lys Val Val Lys Val Ser Ala Ile Pro Phe Arg Ala Pro1505 1510 1515 ttt ttt ccc acc gga gtg aaa gtt gat ccc gag tgc agg atcgtg 4781 Phe Phe Pro Thr Gly Val Lys Val Asp Pro Glu Cys Arg Ile Val1520 1525 1530 gtc gac ccc gat act ttt act aca gcc ctc cgg tct ggt tactct 4826 Val Asp Pro Asp Thr Phe Thr Thr Ala Leu Arg Ser Gly Tyr Ser1535 1540 1545 acc aca aac ctc gtc ctt ggt gtg ggg gac ttt gcc cag ctgaat 4871 Thr Thr Asn Leu Val Leu Gly Val Gly Asp Phe Ala Gln Leu Asn1550 1555 1560 gga cta aag atc agg caa att tcc aag cct tcg gga gga ggccca 4916 Gly Leu Lys Ile Arg Gln Ile Ser Lys Pro Ser Gly Gly Gly Pro1565 1570 1575 cac ctc att gct gcc ctg cat gtt gcc tgc tcg atg gcg ttgcac 4961 His Leu Ile Ala Ala Leu His Val Ala Cys Ser Met Ala Leu His1580 1585 1590 atg ctt gct ggg gtt tat gta act tca gtg ggg tct tgc ggtgcc 5006 Met Leu Ala Gly Val Tyr Val Thr Ser Val Gly Ser Cys Gly Ala1595 1600 1605 ggc acc aac gat cca tgg tgc act aat ccg ttt gcc gtt cctggc 5051 Gly Thr Asn Asp Pro Trp Cys Thr Asn Pro Phe Ala Val Pro Gly1610 1615 1620 tac gga cca ggc tct ctc tgc acg tcc aga ttg tgc atc tcccaa 5096 Tyr Gly Pro Gly Ser Leu Cys Thr Ser Arg Leu Cys Ile Ser Gln1625 1630 1635 cat ggc ctt acc ctg ccc ttg aca gca ctt gtg gcg gga ttcggt 5141 His Gly Leu Thr Leu Pro Leu Thr Ala Leu Val Ala Gly Phe Gly1640 1645 1650 ctt cag gaa atc gcc ttg gtc gtt ttg att ttc gtt tcc atcgga 5186 Leu Gln Glu Ile Ala Leu Val Val Leu Ile Phe Val Ser Ile Gly1655 1660 1665 ggc atg gct cat agg ttg agt tgt aag gct gat atg ctg tgcatc 5231 Gly Met Ala His Arg Leu Ser Cys Lys Ala Asp Met Leu Cys Ile1670 1675 1680 tta ctt gca atc gcc agc tat gtt tgg gta ccc ctt acc tggttg 5276 Leu Leu Ala Ile Ala Ser Tyr Val Trp Val Pro Leu Thr Trp Leu1685 1690 1695 ctt tgt gtg ttt cct tgt tgg ttg cgc tgg ttc tct ttg cacccc 5321 Leu Cys Val Phe Pro Cys Trp Leu Arg Trp Phe Ser Leu His Pro1700 1705 1710 ctc acc atc cta tgg ttg gtg ttt ttc ttg att tct gta aatatg 5366 Leu Thr Ile Leu Trp Leu Val Phe Phe Leu Ile Ser Val Asn Met1715 1720 1725 cct tcg gga atc ttg gcc gtg gtg tta ttg gtt tct ctt tggctt 5411 Pro Ser Gly Ile Leu Ala Val Val Leu Leu Val Ser Leu Trp Leu1730 1735 1740 ttg gga cgt tat act aac att gct ggt ctt gtc acc ccc tatgat 5456 Leu Gly Arg Tyr Thr Asn Ile Ala Gly Leu Val Thr Pro Tyr Asp1745 1750 1755 att cat cat tac acc agt ggc ccc cgc ggt gtt gcc gcc ttagct 5501 Ile His His Tyr Thr Ser Gly Pro Arg Gly Val Ala Ala Leu Ala1760 1765 1770 acc gca cca gat gga acc tac ttg gct gcc gtc cgc cgc gctgcg 5546 Thr Ala Pro Asp Gly Thr Tyr Leu Ala Ala Val Arg Arg Ala Ala1775 1780 1785 ttg act ggt cgc acc atg ctg ttc acc ccg tct cag ctt gggtcc 5591 Leu Thr Gly Arg Thr Met Leu Phe Thr Pro Ser Gln Leu Gly Ser1790 1795 1800 ctt ctt gag ggc gct ttc aga act cga aag ccc tca ctg aacacc 5636 Leu Leu Glu Gly Ala Phe Arg Thr Arg Lys Pro Ser Leu Asn Thr1805 1810 1815 gtc aat gtg gtt ggg tcc tcc atg ggc tct ggt gga gtg ttcacc 5681 Val Asn Val Val Gly Ser Ser Met Gly Ser Gly Gly Val Phe Thr1820 1825 1830 atc gac ggg aaa att agg tgc gtg act gcc gca cat gtc cttacg 5726 Ile Asp Gly Lys Ile Arg Cys Val Thr Ala Ala His Val Leu Thr1835 1840 1845 ggt aat tcg gct agg gtt tcc gga gtc ggc ttc aat caa atgctt 5771 Gly Asn Ser Ala Arg Val Ser Gly Val Gly Phe Asn Gln Met Leu1850 1855 1860 gac ttt gat gtg aaa ggg gac ttc gcc ata gct gat tgc ccgaat 5816 Asp Phe Asp Val Lys Gly Asp Phe Ala Ile Ala Asp Cys Pro Asn1865 1870 1875 tgg caa gga gct gct ccc aag acc caa ttc tgc gag gat ggatgg 5861 Trp Gln Gly Ala Ala Pro Lys Thr Gln Phe Cys Glu Asp Gly Trp1880 1885 1890 gct ggc cgt gcc tat tgg ctg aca tcc tct ggc gtc gaa cccggt 5906 Ala Gly Arg Ala Tyr Trp Leu Thr Ser Ser Gly Val Glu Pro Gly1895 1900 1905 gtt att ggg aat gga ttc gcc ttc tgc ttc acc gcg tgc ggcgat 5951 Val Ile Gly Asn Gly Phe Ala Phe Cys Phe Thr Ala Cys Gly Asp1910 1915 1920 tcc ggg tcc cca gtg atc acc gaa gct ggt gag ctt gtc ggcgtt 5996 Ser Gly Ser Pro Val Ile Thr Glu Ala Gly Glu Leu Val Gly Val1925 1930 1935 cac aca gga tca aat aaa caa gga ggt ggc atc gtc acg cgccct 6041 His Thr Gly Ser Asn Lys Gln Gly Gly Gly Ile Val Thr Arg Pro1940 1945 1950 tca ggc cag ttt tgt aac gtg gca ccc atc aag ctg agc gaatta 6086 Ser Gly Gln Phe Cys Asn Val Ala Pro Ile Lys Leu Ser Glu Leu1955 1960 1965 agt gaa ttc ttt gct gga ccc aag gtc ccg ctc ggt gat gtgaag 6131 Ser Glu Phe Phe Ala Gly Pro Lys Val Pro Leu Gly Asp Val Lys1970 1975 1980 gtt ggc agc cac ata att aaa gat acg tgc gaa gta cct tcagat 6176 Val Gly Ser His Ile Ile Lys Asp Thr Cys Glu Val Pro Ser Asp1985 1990 1995 ctt tgc gcc ttg ctt gct gcc aaa cct gaa ctg gag gga ggcctc 6221 Leu Cys Ala Leu Leu Ala Ala Lys Pro Glu Leu Glu Gly Gly Leu2000 2005 2010 tcc acc gtc caa ctt ctg tgt gtg ttt ttc cta ctg tgg agaatg 6266 Ser Thr Val Gln Leu Leu Cys Val Phe Phe Leu Leu Trp Arg Met2015 2020 2025 atg gga cat gcc tgg acg ccc ttg gtt gct gtg ggg ttt ttcatt 6311 Met Gly His Ala Trp Thr Pro Leu Val Ala Val Gly Phe Phe Ile2030 2035 2040 ctg aat gag gtt ctc cca gct gtc ctg gtt cgg agt gtt ttctcc 6356 Leu Asn Glu Val Leu Pro Ala Val Leu Val Arg Ser Val Phe Ser2045 2050 2055 ttt ggg atg ttt gtg cta tct tgg ctc aca cca tgg tct gcgcaa 6401 Phe Gly Met Phe Val Leu Ser Trp Leu Thr Pro Trp Ser Ala Gln2060 2065 2070 gtt ctg atg atc agg ctt cta aca gca gct ctt aac agg aacaga 6446 Val Leu Met Ile Arg Leu Leu Thr Ala Ala Leu Asn Arg Asn Arg2075 2080 2085 tgg tca ctt gcc ttt tac agc ctt ggt gcg gtg acc ggt tttgtc 6491 Trp Ser Leu Ala Phe Tyr Ser Leu Gly Ala Val Thr Gly Phe Val2090 2095 2100 gca gat ctt gcg gca act caa ggg cac ccg ttg cag gca gtaatg 6536 Ala Asp Leu Ala Ala Thr Gln Gly His Pro Leu Gln Ala Val Met2105 2110 2115 aat ttg agc acc tat gcc ttc ctg cct cgg atg atg gtt gtgacc 6581 Asn Leu Ser Thr Tyr Ala Phe Leu Pro Arg Met Met Val Val Thr2120 2125 2130 tca cca gtc cca gtg att gcg tgt ggt gtt gtg cac cta cttgcc 6626 Ser Pro Val Pro Val Ile Ala Cys Gly Val Val His Leu Leu Ala2135 2140 2145 atc att ttg tac ttg ttc aag tac cgc ggc ctg cac aat gttctt 6671 Ile Ile Leu Tyr Leu Phe Lys Tyr Arg Gly Leu His Asn Val Leu2150 2155 2160 gtt ggt gat gga gcg ttt tct gca gct ttc ttc ttg cga tacttt 6716 Val Gly Asp Gly Ala Phe Ser Ala Ala Phe Phe Leu Arg Tyr Phe2165 2170 2175 gcc gag gga aag ttg agg gaa ggg gtg tcg caa tcc tgc ggaatg 6761 Ala Glu Gly Lys Leu Arg Glu Gly Val Ser Gln Ser Cys Gly Met2180 2185 2190 aat cat gag tca tta act ggt gcc ctc gct atg aga ctc aatgac 6806 Asn His Glu Ser Leu Thr Gly Ala Leu Ala Met Arg Leu Asn Asp2195 2200 2205 gag gac ttg gac ttc ctt acg aaa tgg act gat ttt aag tgcttt 6851 Glu Asp Leu Asp Phe Leu Thr Lys Trp Thr Asp Phe Lys Cys Phe2210 2215 2220 gtt tct gcg tcc aac atg agg aat gca gca ggc caa ttc atcgag 6896 Val Ser Ala Ser Asn Met Arg Asn Ala Ala Gly Gln Phe Ile Glu2225 2230 2235 gct gcc tat gca aaa gca ctt aga att gaa ctt gcc cag ttggtg 6941 Ala Ala Tyr Ala Lys Ala Leu Arg Ile Glu Leu Ala Gln Leu Val2240 2245 2250 cag gtt gat aag gtt cga ggt act ttg gcc aag ctt gag gctttt 6986 Gln Val Asp Lys Val Arg Gly Thr Leu Ala Lys Leu Glu Ala Phe2255 2260 2265 gct gat acc gtg gca ccc caa ctc tcg ccc ggt gac att gttgtt 7031 Ala Asp Thr Val Ala Pro Gln Leu Ser Pro Gly Asp Ile Val Val2270 2275 2280 gct ctt ggc cat acg cct gtt ggc agc atc ttc gac cta aaggtt 7076 Ala Leu Gly His Thr Pro Val Gly Ser Ile Phe Asp Leu Lys Val2285 2290 2295 ggt ggt acc aag cat act ctc caa gtc att gag acc aga gtcctt 7121 Gly Gly Thr Lys His Thr Leu Gln Val Ile Glu Thr Arg Val Leu2300 2305 2310 gcc ggg tcc aaa atg acc gtg gcg cgc gtc gtt gac cca accccc 7166 Ala Gly Ser Lys Met Thr Val Ala Arg Val Val Asp Pro Thr Pro2315 2320 2325 acg ccc cca ccc gca ccc gtg ccc atc ccc ctc cca ccg aaagtt 7211 Thr Pro Pro Pro Ala Pro Val Pro Ile Pro Leu Pro Pro Lys Val2330 2335 2340 cta gag aat ggt ccc aac gcc tgg ggg gat ggg gac cgt ttgaat 7256 Leu Glu Asn Gly Pro Asn Ala Trp Gly Asp Gly Asp Arg Leu Asn2345 2350 2355 aag aag aag agg cgt agg atg gaa acc gtc ggc atc ttt gtcatg 7301 Lys Lys Lys Arg Arg Arg Met Glu Thr Val Gly Ile Phe Val Met2360 2365 2370 ggt ggg aag aag tac cag aaa ttt tgg gac aag aat tcc ggtgat 7346 Gly Gly Lys Lys Tyr Gln Lys Phe Trp Asp Lys Asn Ser Gly Asp2375 2380 2385 gtg ttt tac gag gag gtc cat gac aac aca gat gcg tgg gagtgc 7391 Val Phe Tyr Glu Glu Val His Asp Asn Thr Asp Ala Trp Glu Cys2390 2395 2400 ctc aga gtt ggt gac cct gcc gac ttt gac cct gag aag ggaact 7436 Leu Arg Val Gly Asp Pro Ala Asp Phe Asp Pro Glu Lys Gly Thr2405 2410 2415 ctg tgt ggg cat act act att gaa gat aag gat tac aaa gtctac 7481 Leu Cys Gly His Thr Thr Ile Glu Asp Lys Asp Tyr Lys Val Tyr2420 2425 2430 gcc tcc cca tct ggc aag aag ttc ctg gtc ccc gtc aac tcagag 7526 Ala Ser Pro Ser Gly Lys Lys Phe Leu Val Pro Val Asn Ser Glu2435 2440 2445 agc gga aga gcc caa tgg gaa gct gca aag ctt tcc gtg gagcag 7571 Ser Gly Arg Ala Gln Trp Glu Ala Ala Lys Leu Ser Val Glu Gln2450 2455 2460 gcc ctt ggc atg atg aat gtc gac ggt gaa ctg acg gcc aaagaa 7616 Ala Leu Gly Met Met Asn Val Asp Gly Glu Leu Thr Ala Lys Glu2465 2470 2475 gtg gag aaa ctg aaa aga ata att gac aaa ctt cag ggc ctgact 7661 Val Glu Lys Leu Lys Arg Ile Ile Asp Lys Leu Gln Gly Leu Thr2480 2485 2490 aag gag cag tgt tta aac tgc tag ccgccagcgg cttgacccgctgtggtcgcg 7715 Lys Glu Gln Cys Leu Asn Cys 2495 gcggcttggt tgttactgagacagcggtaa aaatagtcaa atttcacaac cggactttca 7775 ccctagggcc tgtgaatttaaaagtggcca gtgaggttga gctgaaagac gcggtcgagc 7835 acaaccaaca cccggttgcaagaccggttg acggtggtgt tgtgctcctg cgttccgcag 7895 ttccttcgct tatagatgtcctgatctccg gtgctgacgc atctcctaag ttactcgctc 7955 gtcacgggcc ggggaacactgggatcgatg gcacgctttg ggactttgag gccgaggcca 8015 ccaaagagga aattgcactcagtgcgcaaa taatacaggc ttgtgacatt aggcgcggcg 8075 acgcacctga aattggtctcccttacaagc tgtaccctgt taggggcaac cctgagcggg 8135 taaaaggagt tttacagaatacaaggtttg gagacatacc ttacaaaacc cccagtgaca 8195 ctggaagccc agtgcacgcggctgcctgcc tcacgcccaa tgccactccg gtgactgatg 8255 ggcgctctgt cttggctactaccatgccct ccggttttga attgtatgta ccgaccattc 8315 cagcgtctgt ccttgattatcttgactcta ggcctgactg ccccaaacag ttgacagagc 8375 acggctgtga ggatgccgcattgagagacc tctccaagta tgacttgtcc acccaaggct 8435 ttgttttacc tggggttcttcgccttgtgc gtaagtacct gtttgcccat gtgggtaagt 8495 gcccgcccgt tcatcggccttccacttacc ctgccaagaa ttctatggct ggaataaatg 8555 ggaacaggtt tccaaccaaggacattcaga gcgtccctga aatcgacgtt ctgtgcgcac 8615 aggccgtgcg agaaaactggcaaactgtta ccccttgtac cctcaagaaa cagtattgtg 8675 ggaagaagaa gactaggacaatactcggca ccaataattt cattgcgttg gcccaccggg 8735 cagcgttgag tggtgtcacccagggcttca tgaaaaaggc gtttaactcg cccatcgccc 8795 tcgggaaaaa caaatttaaggagctacaga ctccggtctt aggcaggtgc cttgaagctg 8855 atcttgcatc ctgtgatcgatccacacctg caattgtccg ctggtttgcc gccaatcttc 8915 tttatgaact tgcctgtgctgaagagcacc taccgtcgta cgtgctgaac tgctgccatg 8975 acctattggt cacgcagtccggcgcagtga ctaagagggg tggcctatcg tctggcgacc 9035 cgatcacttc tgtgtctaacaccatttaca gcttggtgat atatgcacag cacatggtgc 9095 ttagttactt taaaagtggtcaccctcatg gccttctgtt cctacaagac cagctgaagt 9155 tcgaggacat gctcaaagtccaacccctga tcgtctattc ggacgacctc gtgctgtatg 9215 ccgaatctcc caccatgccgaactaccact ggtgggtcga acatctgaat ttgatgctgg 9275 gttttcagac ggacccaaagaagacagcca taacggactc gccatcattt ctaggctgta 9335 ggataataaa tggacgccagctagtcccca accgtgacag gatcctcgcg gccctcgctt 9395 accatatgaa ggcaagcaatgtttctgaat actacgccgc ggcggctgca atactcatgg 9455 acagctgtgc ttgtttagagtatgatcctg aatggtttga agagcttgtg gttgggatag 9515 cgcagtgcgc ccgcaaggacggctacagct ttcccggccc gccgttcttc ttgtccatgt 9575 gggaaaaact cagatccaatcatgagggga agaagtccag aatgtgcggg tattgcgggg 9635 ccccggctcc gtacgccactgcctgtggcc tcgacgtctg tatttaccac acccacttcc 9695 accagcattg tccagtcataatctggtgtg gccacccggc tggttctggt tcttgtagtg 9755 agtgcaaacc ccccctagggaaaggcacaa gccctctaga tgaggtgtta gaacaagtcc 9815 cgtataagcc tccacggactgtaatcatgc atgtggagca gggtctcacc cctcttgacc 9875 caggcagata ccagactcgccgcggattag tctccgttag gcgtggcatc agaggaaacg 9935 aagttgacct accagacggtgattatgcta gcaccgccct actccccact tgtaaagaga 9995 tcaacatggt cgctgtcgcctctaatgtgt tgcgcagcag gttcatcatc ggtccgcccg 10055 gtgctgggaa aacatactggctccttcagc aggtccagga tggtgatgtc atttacacac 10115 cgactcacca gaccatgctcgacatgatta gggctttggg gacgtgccgg ttcaacgtcc 10175 cagcaggtac aacgctgcaattccctgccc cctcccgtac cggcccgtgg gttcgcatcc 10235 tggccggcgg ttggtgtcctggtaagaatt ccttcctgga tgaagcagcg tattgtaatc 10295 accttgatgt cttgaggctccttagcaaaa ccacccttac ctgtctggga gacttcaaac 10355 aactccaccc agtgggttttgattctcatt gctatgtttt tgacatcatg cctcagaccc 10415 agttgaagac catctggagattcggacaga acatctgtga tgccatccaa ccagattaca 10475 gggacaaact tgtgtccatggtcaacacaa cccgtgtaac ctacatggaa aaacctgtca 10535 agtatgggca agtcctcaccccttaccaca gggaccgaga ggacggcgcc atcacaattg 10595 actccagtca aggcgccacatttgatgtgg ttacactgca tttgcccact aaagattcac 10655 tcaacaggca aagagcccttgttgctatca ccagggcaag acatgctatc tttgtgtatg 10715 acccacacag gcaattgcagagcatgtttg atcttcctgc gaagggcaca cccgtcaacc 10775 tcgcagtgca ccgtgatgagcagctgatcg tactggatag aaataataaa gaatgcacag 10835 ttgctcaggc tataggcaacggagataaat tcagggccac cgacaagcgc gttgtagatt 10895 ctctccgcgc catttgtgctgatctggaag ggtcgagctc cccgctcccc aaggtcgcac 10955 acaacttggg attttatttctcacctgatt tgacacagtt tgctaaactc ccggtagacc 11015 ttgcacccca ctggcccgtggtgacaaccc agaacaatga aaagtggccg gatcggctgg 11075 ttgccagcct tcgccctgtccataagtata gccgtgcgtg cattggtgcc ggctatatgg 11135 tgggcccctc ggtgtttctaggcacccctg gggtcgtgtc atactacctc acaaaatttg 11195 tcaagggcga ggctcaagtgcttccggaga cagtcttcag caccggccga attgaggtgg 11255 attgccggga gtatcttgatgacagggagc gagaagttgc tgagtccctc ccacatgcct 11315 tcattggcga cgtcaaaggcaccaccgttg ggggatgtca tcatgtcacc tccaaatacc 11375 ttccgcgctt ccttcccaaggaatcagtcg cggtagtcgg ggtttcgagc cccgggaaag 11435 ccgcaaaagc agtgtgcacattgacggatg tgtacctccc agaccttgag gcctacctcc 11495 acccagagac tcagtctaagtgctggaaag ttatgttgga cttcaaggaa gttcgactga 11555 tggtctggaa agacaagacggcctatttcc aacttgaagg ccgctatttc acctggtatc 11615 agcttgcaag ctacgcctcgtacatccgtg ttcctgtcaa ctccacggtg tatctggacc 11675 cctgcatggg ccctgccctttgcaacagaa gagttgtcgg gtccacccat tggggagctg 11735 acctcgcagt caccccttatgattacggtg ctaaaatcat cttgtctagc gcttaccatg 11795 gtgaaatgcc tcctggatacaagattctgg cgtgcgcgga gttctcgctc gacgacccag 11855 tcaagtacaa acacacctggggttttgaat cggatacagc gtatctgtat gagttcaccg 11915 gaaacggtga ggactgggaggattacaatg atgcgtttcg tgcgcgccag aaagggaaaa 11975 tttataaggc cactgctaccagcatgaagt tttattttcc cccgggcccc gtcattgaac 12035 caactttagg cctgaattgaa atg aaa tgg ggt cta tac aaa gcc tct tcg 12086 Met Lys Trp Gly Leu TyrLys Ala Ser Ser 2500 2505 aca aaa ttg gcc agc ttt ttg tgg atg ctt tcacgg aat ttt tgg 12131 Thr Lys Leu Ala Ser Phe Leu Trp Met Leu Ser ArgAsn Phe Trp 2510 2515 2520 tgt cca ttg ttg ata tca tca tat ttt tgg ccattt tgt ttg gct 12176 Cys Pro Leu Leu Ile Ser Ser Tyr Phe Trp Pro PheCys Leu Ala 2525 2530 2535 tca cca tcg ccg gtt ggc tgg tgg tct ttt gcatca gat tgg ttt 12221 Ser Pro Ser Pro Val Gly Trp Trp Ser Phe Ala SerAsp Trp Phe 2540 2545 2550 gct ccg cgg tat tcc gtg cgc gcc ctg cca ttcacc ctg agc aat 12266 Ala Pro Arg Tyr Ser Val Arg Ala Leu Pro Phe ThrLeu Ser Asn 2555 2560 2565 tac aga aga tcc tat gag gcc ttt ctt tct cagtgc cgg gtg gac 12311 Tyr Arg Arg Ser Tyr Glu Ala Phe Leu Ser Gln CysArg Val Asp 2570 2575 2580 att ccc acc tgg ggg gta aaa cac cct ttg gggatg ttt tgg cac 12356 Ile Pro Thr Trp Gly Val Lys His Pro Leu Gly MetPhe Trp His 2585 2590 2595 cat aag gtg tca acc ctg att gat gaa atg gtgtcg cgt cga atg 12401 His Lys Val Ser Thr Leu Ile Asp Glu Met Val SerArg Arg Met 2600 2605 2610 tac cgc atc atg gaa aaa gca ggg caa gct gcctgg aaa cag gtg 12446 Tyr Arg Ile Met Glu Lys Ala Gly Gln Ala Ala TrpLys Gln Val 2615 2620 2625 gtg agc gag gct acg ctg tct cgc att agt agtttg gat gtg gtg 12491 Val Ser Glu Ala Thr Leu Ser Arg Ile Ser Ser LeuAsp Val Val 2630 2635 2640 gct cat ttt caa cat ctt gcc gcc att gaa gccgag acc tgt aaa 12536 Ala His Phe Gln His Leu Ala Ala Ile Glu Ala GluThr Cys Lys 2645 2650 2655 tat ttg gct tct cga ctg ccc atg cta cac aacctg cgc atg aca 12581 Tyr Leu Ala Ser Arg Leu Pro Met Leu His Asn LeuArg Met Thr 2660 2665 2670 ggg tca aat gta acc ata gtg tat aat agc acttta aat cag gtg 12626 Gly Ser Asn Val Thr Ile Val Tyr Asn Ser Thr LeuAsn Gln Val 2675 2680 2685 ttt gct att ttt cca acc cct ggt tcc cgg ccaaag ctt cat gat 12671 Phe Ala Ile Phe Pro Thr Pro Gly Ser Arg Pro LysLeu His Asp 2690 2695 2700 ttt cag caa tgg cta ata gct gta cat tcc tccata ttt tcc tct 12716 Phe Gln Gln Trp Leu Ile Ala Val His Ser Ser IlePhe Ser Ser 2705 2710 2715 gtt gca gct tct tgt act ctt ttt gtt gtg ctgtgg ttg cgg gtt 12761 Val Ala Ala Ser Cys Thr Leu Phe Val Val Leu TrpLeu Arg Val 2720 2725 2730 cca atg cta cgt act gtt ttt ggt ttc cgc tggtta ggg gca att 12806 Pro Met Leu Arg Thr Val Phe Gly Phe Arg Trp LeuGly Ala Ile 2735 2740 2745 ttt ctt tcg aac tca tgg tga attacacggtgtgtccacct tgcctcaccc 12857 Phe Leu Ser Asn Ser Trp 2750 gacaagcagccgctgaggtc cttgaacccg gtaggtctct ttggtgcagg atagggcatg 12917 accgatgtggggaggacgat cacgacgaac tagggttcat ggttccgcct ggcctctcca 12977 gcgaaagccacttgaccagt gtttacgcct ggttggcgtt cctgtccttc agctacacgg 13037 cccagttccatcccgagata tttgggatag ggaacgtgag tgaagtttat gttgacatca 13097 agcaccaattcatctgcgcc gttcatgacg ggcagaacac caccttgcct cgccatgaca 13157 atatttcagccgtatttcag acctactatc aacatcaggt cgacggcggc aattggtttc 13217 acctaga atggct gcg tcc ctt ctt ttc ctc ttg gtt ggt ttt aaa 13263 Met Ala Ala SerLeu Leu Phe Leu Leu Val Gly Phe Lys 2755 2760 2765 tgt ttc gtg gtt tctcag gcg ttc gcc tgc aag cca tgt ttc agt 13308 Cys Phe Val Val Ser GlnAla Phe Ala Cys Lys Pro Cys Phe Ser 2770 2775 2780 tcg agt ctt tca gacatc aaa acc aac act acc gca gca tca ggc 13353 Ser Ser Leu Ser Asp IleLys Thr Asn Thr Thr Ala Ala Ser Gly 2785 2790 2795 ttt gtt gtc ctc caggac atc agc tgc ctt agg cat ggc gac tcg 13398 Phe Val Val Leu Gln AspIle Ser Cys Leu Arg His Gly Asp Ser 2800 2805 2810 tcc ttt ccg acg attcgc aaa agc tct caa tgc cgc acg gcg ata 13443 Ser Phe Pro Thr Ile ArgLys Ser Ser Gln Cys Arg Thr Ala Ile 2815 2820 2825 ggg aca ccc gtg tatatc acc atc aca gcc aat gtg aca gat gag 13488 Gly Thr Pro Val Tyr IleThr Ile Thr Ala Asn Val Thr Asp Glu 2830 2835 2840 aat tac tta cat tcttct gat ctc ctc atg ctt tct tct tgc ctt 13533 Asn Tyr Leu His Ser SerAsp Leu Leu Met Leu Ser Ser Cys Leu 2845 2850 2855 ttc tat gct tct gagatg agt gaa aag gga ttc aag gtg gtg ttt 13578 Phe Tyr Ala Ser Glu MetSer Glu Lys Gly Phe Lys Val Val Phe 2860 2865 2870 ggc aat gtg tca ggcatc gtg gct gtg tgt gtc aac ttt acc agc 13623 Gly Asn Val Ser Gly IleVal Ala Val Cys Val Asn Phe Thr Ser 2875 2880 2885 tac gtc caa cat gtcaaa gag ttt acc caa cgc tcc ttg gtg gtc 13668 Tyr Val Gln His Val LysGlu Phe Thr Gln Arg Ser Leu Val Val 2890 2895 2900 gat cat gtg cgg ctgctt cat ttc atg aca cct gag acc atg agg 13713 Asp His Val Arg Leu LeuHis Phe Met Thr Pro Glu Thr Met Arg 2905 2910 2915 tgg gca acc gtt ttagcc tgt ctt ttt gcc atc cta ctg gca att 13758 Trp Ala Thr Val Leu AlaCys Leu Phe Ala Ile Leu Leu Ala Ile 2920 2925 2930 tga atgttcaagt atgttg ggg aaa tgc ttg acc gcg ggc tgt tgc tcg 13807 Met Leu Gly Lys CysLeu Thr Ala Gly Cys Cys Ser 2935 2940 cga ttg ctt tct ttg tgg tgt atcgtg ccg ttc tgt ttt gct gtg 13852 Arg Leu Leu Ser Leu Trp Cys Ile ValPro Phe Cys Phe Ala Val 2945 2950 2955 ctc ggc agc gcc aac agc agc agcagc tct cat ttt cag ttg att 13897 Leu Gly Ser Ala Asn Ser Ser Ser SerSer His Phe Gln Leu Ile 2960 2965 2970 tat aac ttg acg cta tgt gag ctgaat ggc aca gat tgg ctg gca 13942 Tyr Asn Leu Thr Leu Cys Glu Leu AsnGly Thr Asp Trp Leu Ala 2975 2980 2985 gaa aaa ttt gat tgg gca gtg gagact ttt gtc atc ttt ccc gtg 13987 Glu Lys Phe Asp Trp Ala Val Glu ThrPhe Val Ile Phe Pro Val 2990 2995 3000 ttg act cac att gtt tcc tat ggtgca ctc acc acc agc cat ttc 14032 Leu Thr His Ile Val Ser Tyr Gly AlaLeu Thr Thr Ser His Phe 3005 3010 3015 ctt gac aca gtt ggt ctg gtt actgtg tcc acc gcc ggg ttt tat 14077 Leu Asp Thr Val Gly Leu Val Thr ValSer Thr Ala Gly Phe Tyr 3020 3025 3030 cac ggg cgg tat gtc ttg agt agcatc tac gcg gtc tgt gct ctg 14122 His Gly Arg Tyr Val Leu Ser Ser IleTyr Ala Val Cys Ala Leu 3035 3040 3045 gct gcg ttg att tgc ttc gtt attagg ctt gcg aag aac tgc atg 14167 Ala Ala Leu Ile Cys Phe Val Ile ArgLeu Ala Lys Asn Cys Met 3050 3055 3060 tcc tgg cgc tac tct tgt acc agatat acc aac ttc ctt ctg gac 14212 Ser Trp Arg Tyr Ser Cys Thr Arg TyrThr Asn Phe Leu Leu Asp 3065 3070 3075 act aag ggc aga ctc tat cgt tggcgg tcg ccc gtt atc ata gaa 14257 Thr Lys Gly Arg Leu Tyr Arg Trp ArgSer Pro Val Ile Ile Glu 3080 3085 3090 aaa ggg ggt aag gtt gag gtc gaaggt cac ctg atc gac ctc aaa 14302 Lys Gly Gly Lys Val Glu Val Glu GlyHis Leu Ile Asp Leu Lys 3095 3100 3105 aga gtt gtg ctt gat ggt tcc gtggca acc cct tta acc aga gtt 14347 Arg Val Val Leu Asp Gly Ser Val AlaThr Pro Leu Thr Arg Val 3110 3115 3120 tca gcg gaa caa tgg ggt cgt ctctag acgacttttg ccatgatagc 14394 Ser Ala Glu Gln Trp Gly Arg Leu 31253130 acggctccac aaaaggtgct tttggcgttt tccattacct acacgccagt aatgatatat14454 gctctaaagg taagtcgcgg ccgactacta gggcttctgc accttttgat ctttctgaat14514 tgtgctttta ccttcgggta catgacattc gagcactttc agagcacaaa tagggtcgcg14574 ctcactatgg gagcagtagt tgcacttctt tggggggtgt actcagccat agaaacctgg14634 aaattcatca cctccagatg ccgtttgtgc ttgctaggcc gcaagtacat tctggcccct14694 gcccaccacg tcgaaagtgc cgcgggcttt catccgattg cggcaaatga taaccacgca14754 tttgtcgtcc ggcgtcccgg ctccactacg gttaacggca cattggtgcc cgggttgaaa14814 agcctcgtgt tgggtggcag aaaagctgtt aaacagggag tggtaaacct tgtcaaat14872 atg cca aat aac aac ggc aag cag caa aag aaa aag aag ggg aat 14917Met Pro Asn Asn Asn Gly Lys Gln Gln Lys Lys Lys Lys Gly Asn 3135 31403145 ggc cag cca gtc aat cag ctg tgc cag atg ctg ggt aaa atc atc 14962Gly Gln Pro Val Asn Gln Leu Cys Gln Met Leu Gly Lys Ile Ile 3150 31553160 gcc cag caa aac cag tcc aga ggc aag gga ccg ggc aag aaa agt 15007Ala Gln Gln Asn Gln Ser Arg Gly Lys Gly Pro Gly Lys Lys Ser 3165 31703175 aag aag aaa aac ccg gag aag ccc cat ttt cct cta gcg acc gaa 15052Lys Lys Lys Asn Pro Glu Lys Pro His Phe Pro Leu Ala Thr Glu 3180 31853190 gat gac gtc agg cat cac ttc acc cct ggt gag cgg caa ttg tgt 15097Asp Asp Val Arg His His Phe Thr Pro Gly Glu Arg Gln Leu Cys 3195 32003205 ctg tcg tcg atc cag act gcc ttt aac cag ggc gct gga act tgt 15142Leu Ser Ser Ile Gln Thr Ala Phe Asn Gln Gly Ala Gly Thr Cys 3210 32153220 acc ctg tca gat tca ggg agg ata agt tac act gtg gag ttt agt 15187Thr Leu Ser Asp Ser Gly Arg Ile Ser Tyr Thr Val Glu Phe Ser 3225 32303235 ttg ccg acg cat cat act gtg cgc ctg atc cgc gtc aca gca tca 15232Leu Pro Thr His His Thr Val Arg Leu Ile Arg Val Thr Ala Ser 3240 32453250 ccc tca gca tga tgggctggca ttctttaggc acctcagtgt cagaattgga 15284Pro Ser Ala agaatgtgtg gtggatggca ctgattgaca ttgtgcctct aagtcacctattcaattagg 15344 gcgaccgtgt gggggtaaaa tttaattggc gagaaccatg cggccgcaattaaaaaaaaa 15404 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa15450 <210> SEQ ID NO 25 <211> LENGTH: 2497 <212> TYPE: PRT <213>ORGANISM: Arterivirus porcine respiratory and reproductive syndromevirus <400> SEQUENCE: 25 Met Ser Gly Ile Leu Asp Arg Cys Thr Cys Thr ProAsn Ala Arg Val 1 5 10 15 Phe Met Ala Glu Gly Gln Val Tyr Cys Thr ArgCys Leu Ser Ala Arg 20 25 30 Ser Leu Leu Pro Leu Asn Leu Gln Val Pro GluLeu Gly Val Leu Gly 35 40 45 Leu Phe Tyr Arg Pro Glu Glu Pro Leu Arg TrpThr Leu Pro Arg Ala 50 55 60 Phe Pro Thr Val Glu Cys Ser Pro Ala Gly AlaCys Trp Leu Ser Ala 65 70 75 80 Ile Phe Pro Ile Ala Arg Met Thr Ser GlyAsn Leu Asn Phe Gln Gln 85 90 95 Arg Met Val Arg Val Ala Ala Glu Ile TyrArg Ala Gly Gln Leu Thr 100 105 110 Pro Ala Val Leu Lys Ala Leu Gln ValTyr Glu Arg Gly Cys Arg Trp 115 120 125 Tyr Pro Ile Val Gly Pro Val ProGly Val Ala Val His Ala Asn Ser 130 135 140 Leu His Val Ser Asp Lys ProPhe Pro Gly Ala Thr His Val Leu Thr 145 150 155 160 Asn Leu Pro Leu ProGln Arg Pro Lys Pro Glu Asp Phe Cys Pro Phe 165 170 175 Glu Cys Ala MetAla Asp Val Tyr Asp Ile Ser His Asp Ala Val Met 180 185 190 Tyr Val AlaArg Gly Lys Val Ser Trp Ala Pro Arg Gly Gly Asp Glu 195 200 205 Val LysPhe Glu Thr Val Pro Glu Glu Leu Lys Leu Ile Ala Asn Arg 210 215 220 LeuHis Ile Ser Phe Pro Pro His His Ala Val Asp Met Ser Glu Phe 225 230 235240 Ala Phe Ile Ala Pro Gly Ser Gly Val Ser Leu Arg Val Glu His Gln 245250 255 His Gly Cys Leu Pro Ala Asp Thr Val Pro Glu Gly Asn Cys Trp Trp260 265 270 Cys Leu Phe Asp Leu Leu Pro Pro Glu Val Gln Asn Lys Glu IleArg 275 280 285 Arg Ala Asn Gln Phe Gly Tyr Gln Thr Lys His Gly Val ProGly Lys 290 295 300 Tyr Leu Gln Arg Arg Leu Gln Val Asn Gly Leu Arg AlaVal Thr Asp 305 310 315 320 Thr Asp Gly Pro Ile Val Val Gln Tyr Phe SerVal Arg Glu Ser Trp 325 330 335 Ile Arg His Phe Arg Leu Ala Glu Glu ProSer Leu Pro Gly Phe Glu 340 345 350 Asp Leu Leu Arg Ile Arg Val Glu ProAsn Thr Ser Pro Leu Gly Gly 355 360 365 Lys Gly Glu Lys Ile Phe Arg PheGly Ser His Lys Trp Tyr Gly Ala 370 375 380 Gly Lys Arg Ala Arg Arg AlaArg Ser Gly Ala Thr Ala Thr Val Ala 385 390 395 400 His Cys Ala Leu ProAla Arg Glu Ala Gln Gln Ala Lys Lys Leu Glu 405 410 415 Val Ala Ser AlaAsn Arg Ala Glu His Leu Lys Tyr Tyr Ser Pro Pro 420 425 430 Ala Asp GlyAsn Cys Gly Trp His Cys Ile Ser Ala Ile Thr Asn Arg 435 440 445 Met ValAsn Ser Lys Phe Glu Thr Thr Leu Pro Glu Arg Val Arg Pro 450 455 460 SerAsp Asp Trp Ala Thr Asp Glu Asp Leu Val Asn Thr Ile Gln Ile 465 470 475480 Leu Arg Leu Pro Ala Ala Leu Asp Arg Asn Gly Ala Cys Ala Gly Ala 485490 495 Lys Tyr Val Leu Lys Leu Glu Gly Glu His Trp Thr Val Ser Val Thr500 505 510 Pro Gly Met Thr Pro Ser Leu Leu Pro Leu Glu Cys Val Gln GlyCys 515 520 525 Cys Glu His Lys Ser Gly Leu Gly Phe Pro Asp Val Val GluVal Ser 530 535 540 Gly Phe Asp Pro Ala Cys Leu Asp Arg Leu Ala Glu IleMet His Leu 545 550 555 560 Pro Ser Ser Val Ile Pro Ala Ala Leu Ala GluMet Ser Asp Asp Phe 565 570 575 Asn Arg Leu Ala Ser Pro Ala Ala Thr ValTrp Thr Val Ser Gln Phe 580 585 590 Phe Ala Arg His Arg Gly Gly Glu HisPro Asp Gln Val Cys Leu Gly 595 600 605 Lys Ile Ile Asn Leu Cys Gln ValIle Glu Glu Cys Cys Cys Ser Arg 610 615 620 Asn Lys Ala Asn Arg Ala ThrPro Glu Glu Val Ala Ala Lys Val Asp 625 630 635 640 Gln Tyr Leu Arg GlyAla Ala Ser Leu Gly Glu Cys Leu Ala Lys Leu 645 650 655 Glu Arg Ala ArgPro Pro Ser Ala Met Asp Thr Ser Phe Asp Trp Asn 660 665 670 Val Val LeuPro Gly Val Glu Thr Ala Asp Gln Thr Thr Lys Gln Leu 675 680 685 His ValAsn Gln Cys Arg Ala Leu Val Pro Val Val Thr Gln Glu Pro 690 695 700 LeuAsp Arg Asp Ser Val Pro Leu Thr Ala Phe Ser Leu Ser Asn Cys 705 710 715720 Tyr Tyr Pro Ala Gln Gly Asp Glu Val Arg His Arg Glu Arg Leu Asn 725730 735 Ser Val Leu Ser Lys Leu Glu Gly Val Val Arg Glu Glu Tyr Gly Leu740 745 750 Thr Pro Thr Gly Pro Gly Pro Arg Pro Ala Leu Pro Asn Gly LeuAsp 755 760 765 Glu Leu Lys Asp Gln Met Glu Glu Asp Leu Leu Lys Leu ValAsn Ala 770 775 780 Gln Ala Thr Ser Glu Met Met Ala Trp Ala Ala Glu GlnVal Asp Leu 785 790 795 800 Lys Ala Trp Val Lys Asn Tyr Pro Arg Trp ThrPro Pro Pro Pro Pro 805 810 815 Pro Arg Val Gln Pro Arg Lys Thr Lys SerVal Lys Ser Leu Leu Glu 820 825 830 Asn Lys Pro Val Pro Ala Pro Arg ArgLys Val Arg Ser Asp Tyr Gly 835 840 845 Ser Pro Ile Leu Met Gly Asp AsnVal Pro Asn Gly Trp Glu Asp Ser 850 855 860 Thr Val Gly Gly Pro Leu AspLeu Ser Ala Pro Ser Glu Pro Met Thr 865 870 875 880 Pro Leu Ser Glu ProVal Leu Ile Ser Arg Pro Val Thr Ser Leu Ser 885 890 895 Val Pro Ala ProVal Pro Ala Pro Arg Arg Ala Val Ser Arg Pro Met 900 905 910 Thr Pro SerSer Glu Pro Ile Phe Val Ser Ala Leu Arg His Lys Phe 915 920 925 Gln GlnVal Glu Lys Ala Asn Leu Ala Ala Ala Ala Pro Met Tyr Gln 930 935 940 AspGlu Pro Leu Asp Leu Ser Ala Ser Ser Gln Thr Glu Tyr Gly Ala 945 950 955960 Ser Pro Leu Thr Pro Pro Gln Asn Val Gly Ile Leu Glu Val Arg Gly 965970 975 Gln Glu Ala Glu Glu Val Leu Ser Glu Ile Ser Asp Ile Leu Asn Asp980 985 990 Thr Asn Pro Ala Pro Val Ser Ser Ser Ser Ser Leu Ser Ser ValArg 995 1000 1005 Ile Thr Arg Pro Lys Tyr Ser Ala Gln Ala Ile Ile AspLeu Gly 1010 1015 1020 Gly Pro Cys Ser Gly His Leu Gln Arg Glu Lys GluAla Cys Leu 1025 1030 1035 Arg Ile Met Arg Glu Ala Cys Asp Ala Ala LysLeu Ser Asp Pro 1040 1045 1050 Ala Thr Gln Glu Trp Leu Ser Arg Met TrpAsp Arg Val Asp Met 1055 1060 1065 Leu Thr Trp Arg Asn Thr Ser Ala TyrGln Ala Phe Arg Thr Leu 1070 1075 1080 Asp Gly Arg Phe Gly Phe Leu ProLys Met Ile Leu Glu Thr Pro 1085 1090 1095 Pro Pro Tyr Pro Cys Gly PheVal Met Leu Pro His Thr Pro Ala 1100 1105 1110 Pro Ser Val Ser Ala GluSer Asp Leu Thr Ile Gly Ser Val Ala 1115 1120 1125 Thr Glu Asp Ile ProArg Ile Leu Gly Lys Ile Glu Asn Thr Gly 1130 1135 1140 Glu Met Ile AsnGln Gly Pro Leu Ala Ser Ser Glu Glu Glu Pro 1145 1150 1155 Val Tyr AsnGln Pro Ala Lys Asp Ser Arg Ile Ser Ser Arg Gly 1160 1165 1170 Ser AspGlu Ser Thr Ala Ala Pro Ser Ala Gly Thr Gly Gly Ala 1175 1180 1185 GlyLeu Phe Thr Asp Leu Pro Pro Ser Asp Gly Val Asp Ala Asp 1190 1195 1200Gly Gly Gly Pro Leu Gln Thr Val Arg Lys Lys Ala Glu Arg Leu 1205 12101215 Phe Asp Gln Leu Ser Arg Gln Val Phe Asn Leu Val Ser His Leu 12201225 1230 Pro Val Phe Phe Ser His Leu Phe Lys Ser Asp Ser Gly Tyr Ser1235 1240 1245 Pro Gly Asp Trp Gly Phe Ala Ala Phe Thr Leu Phe Cys LeuPhe 1250 1255 1260 Leu Cys Tyr Ser Tyr Pro Phe Phe Gly Phe Val Pro LeuLeu Gly 1265 1270 1275 Val Phe Ser Gly Ser Ser Arg Arg Val Arg Met GlyVal Phe Gly 1280 1285 1290 Cys Trp Leu Ala Phe Ala Val Gly Leu Phe LysPro Val Ser Asp 1295 1300 1305 Pro Val Gly Thr Ala Cys Glu Phe Asp SerPro Glu Cys Arg Asn 1310 1315 1320 Val Leu His Ser Phe Glu Leu Leu LysPro Trp Asp Pro Val Arg 1325 1330 1335 Ser Leu Val Val Gly Pro Val GlyLeu Gly Leu Ala Ile Leu Gly 1340 1345 1350 Arg Leu Leu Gly Gly Ala ArgTyr Ile Trp His Phe Leu Leu Arg 1355 1360 1365 Leu Gly Ile Val Ala AspCys Ile Leu Ala Gly Ala Tyr Val Leu 1370 1375 1380 Ser Gln Gly Arg CysLys Lys Cys Trp Gly Ser Cys Ile Arg Thr 1385 1390 1395 Ala Pro Asn GluIle Ala Phe Asn Val Phe Pro Phe Thr Arg Ala 1400 1405 1410 Thr Arg SerSer Leu Ile Asp Leu Cys Asp Arg Phe Cys Ala Pro 1415 1420 1425 Lys GlyMet Asp Pro Ile Phe Leu Ala Thr Gly Trp Arg Gly Cys 1430 1435 1440 TrpThr Gly Arg Ser Pro Ile Glu Gln Pro Ser Glu Lys Pro Ile 1445 1450 1455Ala Phe Ala Gln Leu Asp Glu Lys Arg Ile Thr Ala Arg Thr Val 1460 14651470 Val Ala Gln Pro Tyr Asp Pro Asn Gln Ala Val Lys Cys Leu Arg 14751480 1485 Val Leu Gln Ala Gly Gly Ala Met Val Ala Glu Ala Val Pro Lys1490 1495 1500 Val Val Lys Val Ser Ala Ile Pro Phe Arg Ala Pro Phe PhePro 1505 1510 1515 Thr Gly Val Lys Val Asp Pro Glu Cys Arg Ile Val ValAsp Pro 1520 1525 1530 Asp Thr Phe Thr Thr Ala Leu Arg Ser Gly Tyr SerThr Thr Asn 1535 1540 1545 Leu Val Leu Gly Val Gly Asp Phe Ala Gln LeuAsn Gly Leu Lys 1550 1555 1560 Ile Arg Gln Ile Ser Lys Pro Ser Gly GlyGly Pro His Leu Ile 1565 1570 1575 Ala Ala Leu His Val Ala Cys Ser MetAla Leu His Met Leu Ala 1580 1585 1590 Gly Val Tyr Val Thr Ser Val GlySer Cys Gly Ala Gly Thr Asn 1595 1600 1605 Asp Pro Trp Cys Thr Asn ProPhe Ala Val Pro Gly Tyr Gly Pro 1610 1615 1620 Gly Ser Leu Cys Thr SerArg Leu Cys Ile Ser Gln His Gly Leu 1625 1630 1635 Thr Leu Pro Leu ThrAla Leu Val Ala Gly Phe Gly Leu Gln Glu 1640 1645 1650 Ile Ala Leu ValVal Leu Ile Phe Val Ser Ile Gly Gly Met Ala 1655 1660 1665 His Arg LeuSer Cys Lys Ala Asp Met Leu Cys Ile Leu Leu Ala 1670 1675 1680 Ile AlaSer Tyr Val Trp Val Pro Leu Thr Trp Leu Leu Cys Val 1685 1690 1695 PhePro Cys Trp Leu Arg Trp Phe Ser Leu His Pro Leu Thr Ile 1700 1705 1710Leu Trp Leu Val Phe Phe Leu Ile Ser Val Asn Met Pro Ser Gly 1715 17201725 Ile Leu Ala Val Val Leu Leu Val Ser Leu Trp Leu Leu Gly Arg 17301735 1740 Tyr Thr Asn Ile Ala Gly Leu Val Thr Pro Tyr Asp Ile His His1745 1750 1755 Tyr Thr Ser Gly Pro Arg Gly Val Ala Ala Leu Ala Thr AlaPro 1760 1765 1770 Asp Gly Thr Tyr Leu Ala Ala Val Arg Arg Ala Ala LeuThr Gly 1775 1780 1785 Arg Thr Met Leu Phe Thr Pro Ser Gln Leu Gly SerLeu Leu Glu 1790 1795 1800 Gly Ala Phe Arg Thr Arg Lys Pro Ser Leu AsnThr Val Asn Val 1805 1810 1815 Val Gly Ser Ser Met Gly Ser Gly Gly ValPhe Thr Ile Asp Gly 1820 1825 1830 Lys Ile Arg Cys Val Thr Ala Ala HisVal Leu Thr Gly Asn Ser 1835 1840 1845 Ala Arg Val Ser Gly Val Gly PheAsn Gln Met Leu Asp Phe Asp 1850 1855 1860 Val Lys Gly Asp Phe Ala IleAla Asp Cys Pro Asn Trp Gln Gly 1865 1870 1875 Ala Ala Pro Lys Thr GlnPhe Cys Glu Asp Gly Trp Ala Gly Arg 1880 1885 1890 Ala Tyr Trp Leu ThrSer Ser Gly Val Glu Pro Gly Val Ile Gly 1895 1900 1905 Asn Gly Phe AlaPhe Cys Phe Thr Ala Cys Gly Asp Ser Gly Ser 1910 1915 1920 Pro Val IleThr Glu Ala Gly Glu Leu Val Gly Val His Thr Gly 1925 1930 1935 Ser AsnLys Gln Gly Gly Gly Ile Val Thr Arg Pro Ser Gly Gln 1940 1945 1950 PheCys Asn Val Ala Pro Ile Lys Leu Ser Glu Leu Ser Glu Phe 1955 1960 1965Phe Ala Gly Pro Lys Val Pro Leu Gly Asp Val Lys Val Gly Ser 1970 19751980 His Ile Ile Lys Asp Thr Cys Glu Val Pro Ser Asp Leu Cys Ala 19851990 1995 Leu Leu Ala Ala Lys Pro Glu Leu Glu Gly Gly Leu Ser Thr Val2000 2005 2010 Gln Leu Leu Cys Val Phe Phe Leu Leu Trp Arg Met Met GlyHis 2015 2020 2025 Ala Trp Thr Pro Leu Val Ala Val Gly Phe Phe Ile LeuAsn Glu 2030 2035 2040 Val Leu Pro Ala Val Leu Val Arg Ser Val Phe SerPhe Gly Met 2045 2050 2055 Phe Val Leu Ser Trp Leu Thr Pro Trp Ser AlaGln Val Leu Met 2060 2065 2070 Ile Arg Leu Leu Thr Ala Ala Leu Asn ArgAsn Arg Trp Ser Leu 2075 2080 2085 Ala Phe Tyr Ser Leu Gly Ala Val ThrGly Phe Val Ala Asp Leu 2090 2095 2100 Ala Ala Thr Gln Gly His Pro LeuGln Ala Val Met Asn Leu Ser 2105 2110 2115 Thr Tyr Ala Phe Leu Pro ArgMet Met Val Val Thr Ser Pro Val 2120 2125 2130 Pro Val Ile Ala Cys GlyVal Val His Leu Leu Ala Ile Ile Leu 2135 2140 2145 Tyr Leu Phe Lys TyrArg Gly Leu His Asn Val Leu Val Gly Asp 2150 2155 2160 Gly Ala Phe SerAla Ala Phe Phe Leu Arg Tyr Phe Ala Glu Gly 2165 2170 2175 Lys Leu ArgGlu Gly Val Ser Gln Ser Cys Gly Met Asn His Glu 2180 2185 2190 Ser LeuThr Gly Ala Leu Ala Met Arg Leu Asn Asp Glu Asp Leu 2195 2200 2205 AspPhe Leu Thr Lys Trp Thr Asp Phe Lys Cys Phe Val Ser Ala 2210 2215 2220Ser Asn Met Arg Asn Ala Ala Gly Gln Phe Ile Glu Ala Ala Tyr 2225 22302235 Ala Lys Ala Leu Arg Ile Glu Leu Ala Gln Leu Val Gln Val Asp 22402245 2250 Lys Val Arg Gly Thr Leu Ala Lys Leu Glu Ala Phe Ala Asp Thr2255 2260 2265 Val Ala Pro Gln Leu Ser Pro Gly Asp Ile Val Val Ala LeuGly 2270 2275 2280 His Thr Pro Val Gly Ser Ile Phe Asp Leu Lys Val GlyGly Thr 2285 2290 2295 Lys His Thr Leu Gln Val Ile Glu Thr Arg Val LeuAla Gly Ser 2300 2305 2310 Lys Met Thr Val Ala Arg Val Val Asp Pro ThrPro Thr Pro Pro 2315 2320 2325 Pro Ala Pro Val Pro Ile Pro Leu Pro ProLys Val Leu Glu Asn 2330 2335 2340 Gly Pro Asn Ala Trp Gly Asp Gly AspArg Leu Asn Lys Lys Lys 2345 2350 2355 Arg Arg Arg Met Glu Thr Val GlyIle Phe Val Met Gly Gly Lys 2360 2365 2370 Lys Tyr Gln Lys Phe Trp AspLys Asn Ser Gly Asp Val Phe Tyr 2375 2380 2385 Glu Glu Val His Asp AsnThr Asp Ala Trp Glu Cys Leu Arg Val 2390 2395 2400 Gly Asp Pro Ala AspPhe Asp Pro Glu Lys Gly Thr Leu Cys Gly 2405 2410 2415 His Thr Thr IleGlu Asp Lys Asp Tyr Lys Val Tyr Ala Ser Pro 2420 2425 2430 Ser Gly LysLys Phe Leu Val Pro Val Asn Ser Glu Ser Gly Arg 2435 2440 2445 Ala GlnTrp Glu Ala Ala Lys Leu Ser Val Glu Gln Ala Leu Gly 2450 2455 2460 MetMet Asn Val Asp Gly Glu Leu Thr Ala Lys Glu Val Glu Lys 2465 2470 2475Leu Lys Arg Ile Ile Asp Lys Leu Gln Gly Leu Thr Lys Glu Gln 2480 24852490 Cys Leu Asn Cys 2495 <210> SEQ ID NO 26 <211> LENGTH: 256 <212>TYPE: PRT <213> ORGANISM: Arterivirus porcine respiratory andreproductive syndrome virus <400> SEQUENCE: 26 Met Lys Trp Gly Leu TyrLys Ala Ser Ser Thr Lys Leu Ala Ser Phe 1 5 10 15 Leu Trp Met Leu SerArg Asn Phe Trp Cys Pro Leu Leu Ile Ser Ser 20 25 30 Tyr Phe Trp Pro PheCys Leu Ala Ser Pro Ser Pro Val Gly Trp Trp 35 40 45 Ser Phe Ala Ser AspTrp Phe Ala Pro Arg Tyr Ser Val Arg Ala Leu 50 55 60 Pro Phe Thr Leu SerAsn Tyr Arg Arg Ser Tyr Glu Ala Phe Leu Ser 65 70 75 80 Gln Cys Arg ValAsp Ile Pro Thr Trp Gly Val Lys His Pro Leu Gly 85 90 95 Met Phe Trp HisHis Lys Val Ser Thr Leu Ile Asp Glu Met Val Ser 100 105 110 Arg Arg MetTyr Arg Ile Met Glu Lys Ala Gly Gln Ala Ala Trp Lys 115 120 125 Gln ValVal Ser Glu Ala Thr Leu Ser Arg Ile Ser Ser Leu Asp Val 130 135 140 ValAla His Phe Gln His Leu Ala Ala Ile Glu Ala Glu Thr Cys Lys 145 150 155160 Tyr Leu Ala Ser Arg Leu Pro Met Leu His Asn Leu Arg Met Thr Gly 165170 175 Ser Asn Val Thr Ile Val Tyr Asn Ser Thr Leu Asn Gln Val Phe Ala180 185 190 Ile Phe Pro Thr Pro Gly Ser Arg Pro Lys Leu His Asp Phe GlnGln 195 200 205 Trp Leu Ile Ala Val His Ser Ser Ile Phe Ser Ser Val AlaAla Ser 210 215 220 Cys Thr Leu Phe Val Val Leu Trp Leu Arg Val Pro MetLeu Arg Thr 225 230 235 240 Val Phe Gly Phe Arg Trp Leu Gly Ala Ile PheLeu Ser Asn Ser Trp 245 250 255 <210> SEQ ID NO 27 <211> LENGTH: 178<212> TYPE: PRT <213> ORGANISM: Arterivirus porcine respiratory andreproductive syndrome virus <400> SEQUENCE: 27 Met Ala Ala Ser Leu LeuPhe Leu Leu Val Gly Phe Lys Cys Phe Val 1 5 10 15 Val Ser Gln Ala PheAla Cys Lys Pro Cys Phe Ser Ser Ser Leu Ser 20 25 30 Asp Ile Lys Thr AsnThr Thr Ala Ala Ser Gly Phe Val Val Leu Gln 35 40 45 Asp Ile Ser Cys LeuArg His Gly Asp Ser Ser Phe Pro Thr Ile Arg 50 55 60 Lys Ser Ser Gln CysArg Thr Ala Ile Gly Thr Pro Val Tyr Ile Thr 65 70 75 80 Ile Thr Ala AsnVal Thr Asp Glu Asn Tyr Leu His Ser Ser Asp Leu 85 90 95 Leu Met Leu SerSer Cys Leu Phe Tyr Ala Ser Glu Met Ser Glu Lys 100 105 110 Gly Phe LysVal Val Phe Gly Asn Val Ser Gly Ile Val Ala Val Cys 115 120 125 Val AsnPhe Thr Ser Tyr Val Gln His Val Lys Glu Phe Thr Gln Arg 130 135 140 SerLeu Val Val Asp His Val Arg Leu Leu His Phe Met Thr Pro Glu 145 150 155160 Thr Met Arg Trp Ala Thr Val Leu Ala Cys Leu Phe Ala Ile Leu Leu 165170 175 Ala Ile <210> SEQ ID NO 28 <211> LENGTH: 200 <212> TYPE: PRT<213> ORGANISM: Arterivirus porcine respiratory and reproductivesyndrome virus <400> SEQUENCE: 28 Met Leu Gly Lys Cys Leu Thr Ala GlyCys Cys Ser Arg Leu Leu Ser 1 5 10 15 Leu Trp Cys Ile Val Pro Phe CysPhe Ala Val Leu Gly Ser Ala Asn 20 25 30 Ser Ser Ser Ser Ser His Phe GlnLeu Ile Tyr Asn Leu Thr Leu Cys 35 40 45 Glu Leu Asn Gly Thr Asp Trp LeuAla Glu Lys Phe Asp Trp Ala Val 50 55 60 Glu Thr Phe Val Ile Phe Pro ValLeu Thr His Ile Val Ser Tyr Gly 65 70 75 80 Ala Leu Thr Thr Ser His PheLeu Asp Thr Val Gly Leu Val Thr Val 85 90 95 Ser Thr Ala Gly Phe Tyr HisGly Arg Tyr Val Leu Ser Ser Ile Tyr 100 105 110 Ala Val Cys Ala Leu AlaAla Leu Ile Cys Phe Val Ile Arg Leu Ala 115 120 125 Lys Asn Cys Met SerTrp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe 130 135 140 Leu Leu Asp ThrLys Gly Arg Leu Tyr Arg Trp Arg Ser Pro Val Ile 145 150 155 160 Ile GluLys Gly Gly Lys Val Glu Val Glu Gly His Leu Ile Asp Leu 165 170 175 LysArg Val Val Leu Asp Gly Ser Val Ala Thr Pro Leu Thr Arg Val 180 185 190Ser Ala Glu Gln Trp Gly Arg Leu 195 200 <210> SEQ ID NO 29 <211> LENGTH:123 <212> TYPE: PRT <213> ORGANISM: Arterivirus porcine respiratory andreproductive syndrome virus <400> SEQUENCE: 29 Met Pro Asn Asn Asn GlyLys Gln Gln Lys Lys Lys Lys Gly Asn Gly 1 5 10 15 Gln Pro Val Asn GlnLeu Cys Gln Met Leu Gly Lys Ile Ile Ala Gln 20 25 30 Gln Asn Gln Ser ArgGly Lys Gly Pro Gly Lys Lys Ser Lys Lys Lys 35 40 45 Asn Pro Glu Lys ProHis Phe Pro Leu Ala Thr Glu Asp Asp Val Arg 50 55 60 His His Phe Thr ProGly Glu Arg Gln Leu Cys Leu Ser Ser Ile Gln 65 70 75 80 Thr Ala Phe AsnGln Gly Ala Gly Thr Cys Thr Leu Ser Asp Ser Gly 85 90 95 Arg Ile Ser TyrThr Val Glu Phe Ser Leu Pro Thr His His Thr Val 100 105 110 Arg Leu IleArg Val Thr Ala Ser Pro Ser Ala 115 120 <210> SEQ ID NO 30 <211> LENGTH:1463 <212> TYPE: PRT <213> ORGANISM: Arterivirus porcine respiratory andreproductive syndrome virus <220> FEATURE: <221> NAME/KEY: MISC_FEATURE<222> LOCATION: (1)..(1457) <223> OTHER INFORMATION: ORF 1b, nucleotides7682 to 12055 of the viral sequence <220> FEATURE: <221> NAME/KEY:MISC_FEATURE <222> LOCATION: (1)..(1457) <223> OTHER INFORMATION: ORF1b, nucleotides 7664 to 12055 of the viral sequence <400> SEQUENCE: 30Gly Ala Val Phe Lys Leu Leu Ala Ala Ser Gly Leu Thr Arg Cys Gly 1 5 1015 Arg Gly Gly Leu Val Val Thr Glu Thr Ala Val Lys Ile Val Lys Phe 20 2530 His Asn Arg Thr Phe Thr Leu Gly Pro Val Asn Leu Lys Val Ala Ser 35 4045 Glu Val Glu Leu Lys Asp Ala Val Glu His Asn Gln His Pro Val Ala 50 5560 Arg Pro Val Asp Gly Gly Val Val Leu Leu Arg Ser Ala Val Pro Ser 65 7075 80 Leu Ile Asp Val Leu Ile Ser Gly Ala Asp Ala Ser Pro Lys Leu Leu 8590 95 Ala Arg His Gly Pro Gly Asn Thr Gly Ile Asp Gly Thr Leu Trp Asp100 105 110 Phe Glu Ala Glu Ala Thr Lys Glu Glu Ile Ala Leu Ser Ala GlnIle 115 120 125 Ile Gln Ala Cys Asp Ile Arg Arg Gly Asp Ala Pro Glu IleGly Leu 130 135 140 Pro Tyr Lys Leu Tyr Pro Val Arg Gly Asn Pro Glu ArgVal Lys Gly 145 150 155 160 Val Leu Gln Asn Thr Arg Phe Gly Asp Ile ProTyr Lys Thr Pro Ser 165 170 175 Asp Thr Gly Ser Pro Val His Ala Ala AlaCys Leu Thr Pro Asn Ala 180 185 190 Thr Pro Val Thr Asp Gly Arg Ser ValLeu Ala Thr Thr Met Pro Ser 195 200 205 Gly Phe Glu Leu Tyr Val Pro ThrIle Pro Ala Ser Val Leu Asp Tyr 210 215 220 Leu Asp Ser Arg Pro Asp CysPro Lys Gln Leu Thr Glu His Gly Cys 225 230 235 240 Glu Asp Ala Ala LeuArg Asp Leu Ser Lys Tyr Asp Leu Ser Thr Gln 245 250 255 Gly Phe Val LeuPro Gly Val Leu Arg Leu Val Arg Lys Tyr Leu Phe 260 265 270 Ala His ValGly Lys Cys Pro Pro Val His Arg Pro Ser Thr Tyr Pro 275 280 285 Ala LysAsn Ser Met Ala Gly Ile Asn Gly Asn Arg Phe Pro Thr Lys 290 295 300 AspIle Gln Ser Val Pro Glu Ile Asp Val Leu Cys Ala Gln Ala Val 305 310 315320 Arg Glu Asn Trp Gln Thr Val Thr Pro Cys Thr Leu Lys Lys Gln Tyr 325330 335 Cys Gly Lys Lys Lys Thr Arg Thr Ile Leu Gly Thr Asn Asn Phe Ile340 345 350 Ala Leu Ala His Arg Ala Ala Leu Ser Gly Val Thr Gln Gly PheMet 355 360 365 Lys Lys Ala Phe Asn Ser Pro Ile Ala Leu Gly Lys Asn LysPhe Lys 370 375 380 Glu Leu Gln Thr Pro Val Leu Gly Arg Cys Leu Glu AlaAsp Leu Ala 385 390 395 400 Ser Cys Asp Arg Ser Thr Pro Ala Ile Val ArgTrp Phe Ala Ala Asn 405 410 415 Leu Leu Tyr Glu Leu Ala Cys Ala Glu GluHis Leu Pro Ser Tyr Val 420 425 430 Leu Asn Cys Cys His Asp Leu Leu ValThr Gln Ser Gly Ala Val Thr 435 440 445 Lys Arg Gly Gly Leu Ser Ser GlyAsp Pro Ile Thr Ser Val Ser Asn 450 455 460 Thr Ile Tyr Ser Leu Val IleTyr Ala Gln His Met Val Leu Ser Tyr 465 470 475 480 Phe Lys Ser Gly HisPro His Gly Leu Leu Phe Leu Gln Asp Gln Leu 485 490 495 Lys Phe Glu AspMet Leu Lys Val Gln Pro Leu Ile Val Tyr Ser Asp 500 505 510 Asp Leu ValLeu Tyr Ala Glu Ser Pro Thr Met Pro Asn Tyr His Trp 515 520 525 Trp ValGlu His Leu Asn Leu Met Leu Gly Phe Gln Thr Asp Pro Lys 530 535 540 LysThr Ala Ile Thr Asp Ser Pro Ser Phe Leu Gly Cys Arg Ile Ile 545 550 555560 Asn Gly Arg Gln Leu Val Pro Asn Arg Asp Arg Ile Leu Ala Ala Leu 565570 575 Ala Tyr His Met Lys Ala Ser Asn Val Ser Glu Tyr Tyr Ala Ala Ala580 585 590 Ala Ala Ile Leu Met Asp Ser Cys Ala Cys Leu Glu Tyr Asp ProGlu 595 600 605 Trp Phe Glu Glu Leu Val Val Gly Ile Ala Gln Cys Ala ArgLys Asp 610 615 620 Gly Tyr Ser Phe Pro Gly Pro Pro Phe Phe Leu Ser MetTrp Glu Lys 625 630 635 640 Leu Arg Ser Asn His Glu Gly Lys Lys Ser ArgMet Cys Gly Tyr Cys 645 650 655 Gly Ala Pro Ala Pro Tyr Ala Thr Ala CysGly Leu Asp Val Cys Ile 660 665 670 Tyr His Thr His Phe His Gln His CysPro Val Ile Ile Trp Cys Gly 675 680 685 His Pro Ala Gly Ser Gly Ser CysSer Glu Cys Lys Pro Pro Leu Gly 690 695 700 Lys Gly Thr Ser Pro Leu AspGlu Val Leu Glu Gln Val Pro Tyr Lys 705 710 715 720 Pro Pro Arg Thr ValIle Met His Val Glu Gln Gly Leu Thr Pro Leu 725 730 735 Asp Pro Gly ArgTyr Gln Thr Arg Arg Gly Leu Val Ser Val Arg Arg 740 745 750 Gly Ile ArgGly Asn Glu Val Asp Leu Pro Asp Gly Asp Tyr Ala Ser 755 760 765 Thr AlaLeu Leu Pro Thr Cys Lys Glu Ile Asn Met Val Ala Val Ala 770 775 780 SerAsn Val Leu Arg Ser Arg Phe Ile Ile Gly Pro Pro Gly Ala Gly 785 790 795800 Lys Thr Tyr Trp Leu Leu Gln Gln Val Gln Asp Gly Asp Val Ile Tyr 805810 815 Thr Pro Thr His Gln Thr Met Leu Asp Met Ile Arg Ala Leu Gly Thr820 825 830 Cys Arg Phe Asn Val Pro Ala Gly Thr Thr Leu Gln Phe Pro AlaPro 835 840 845 Ser Arg Thr Gly Pro Trp Val Arg Ile Leu Ala Gly Gly TrpCys Pro 850 855 860 Gly Lys Asn Ser Phe Leu Asp Glu Ala Ala Tyr Cys AsnHis Leu Asp 865 870 875 880 Val Leu Arg Leu Leu Ser Lys Thr Thr Leu ThrCys Leu Gly Asp Phe 885 890 895 Lys Gln Leu His Pro Val Gly Phe Asp SerHis Cys Tyr Val Phe Asp 900 905 910 Ile Met Pro Gln Thr Gln Leu Lys ThrIle Trp Arg Phe Gly Gln Asn 915 920 925 Ile Cys Asp Ala Ile Gln Pro AspTyr Arg Asp Lys Leu Val Ser Met 930 935 940 Val Asn Thr Thr Arg Val ThrTyr Met Glu Lys Pro Val Lys Tyr Gly 945 950 955 960 Gln Val Leu Thr ProTyr His Arg Asp Arg Glu Asp Gly Ala Ile Thr 965 970 975 Ile Asp Ser SerGln Gly Ala Thr Phe Asp Val Val Thr Leu His Leu 980 985 990 Pro Thr LysAsp Ser Leu Asn Arg Gln Arg Ala Leu Val Ala Ile Thr 995 1000 1005 ArgAla Arg His Ala Ile Phe Val Tyr Asp Pro His Arg Gln Leu 1010 1015 1020Gln Ser Met Phe Asp Leu Pro Ala Lys Gly Thr Pro Val Asn Leu 1025 10301035 Ala Val His Arg Asp Glu Gln Leu Ile Val Leu Asp Arg Asn Asn 10401045 1050 Lys Glu Cys Thr Val Ala Gln Ala Ile Gly Asn Gly Asp Lys Phe1055 1060 1065 Arg Ala Thr Asp Lys Arg Val Val Asp Ser Leu Arg Ala IleCys 1070 1075 1080 Ala Asp Leu Glu Gly Ser Ser Ser Pro Leu Pro Lys ValAla His 1085 1090 1095 Asn Leu Gly Phe Tyr Phe Ser Pro Asp Leu Thr GlnPhe Ala Lys 1100 1105 1110 Leu Pro Val Asp Leu Ala Pro His Trp Pro ValVal Thr Thr Gln 1115 1120 1125 Asn Asn Glu Lys Trp Pro Asp Arg Leu ValAla Ser Leu Arg Pro 1130 1135 1140 Val His Lys Tyr Ser Arg Ala Cys IleGly Ala Gly Tyr Met Val 1145 1150 1155 Gly Pro Ser Val Phe Leu Gly ThrPro Gly Val Val Ser Tyr Tyr 1160 1165 1170 Leu Thr Lys Phe Val Lys GlyGlu Ala Gln Val Leu Pro Glu Thr 1175 1180 1185 Val Phe Ser Thr Gly ArgIle Glu Val Asp Cys Arg Glu Tyr Leu 1190 1195 1200 Asp Asp Arg Glu ArgGlu Val Ala Glu Ser Leu Pro His Ala Phe 1205 1210 1215 Ile Gly Asp ValLys Gly Thr Thr Val Gly Gly Cys His His Val 1220 1225 1230 Thr Ser LysTyr Leu Pro Arg Phe Leu Pro Lys Glu Ser Val Ala 1235 1240 1245 Val ValGly Val Ser Ser Pro Gly Lys Ala Ala Lys Ala Val Cys 1250 1255 1260 ThrLeu Thr Asp Val Tyr Leu Pro Asp Leu Glu Ala Tyr Leu His 1265 1270 1275Pro Glu Thr Gln Ser Lys Cys Trp Lys Val Met Leu Asp Phe Lys 1280 12851290 Glu Val Arg Leu Met Val Trp Lys Asp Lys Thr Ala Tyr Phe Gln 12951300 1305 Leu Glu Gly Arg Tyr Phe Thr Trp Tyr Gln Leu Ala Ser Tyr Ala1310 1315 1320 Ser Tyr Ile Arg Val Pro Val Asn Ser Thr Val Tyr Leu AspPro 1325 1330 1335 Cys Met Gly Pro Ala Leu Cys Asn Arg Arg Val Val GlySer Thr 1340 1345 1350 His Trp Gly Ala Asp Leu Ala Val Thr Pro Tyr AspTyr Gly Ala 1355 1360 1365 Lys Ile Ile Leu Ser Ser Ala Tyr His Gly GluMet Pro Pro Gly 1370 1375 1380 Tyr Lys Ile Leu Ala Cys Ala Glu Phe SerLeu Asp Asp Pro Val 1385 1390 1395 Lys Tyr Lys His Thr Trp Gly Phe GluSer Asp Thr Ala Tyr Leu 1400 1405 1410 Tyr Glu Phe Thr Gly Asn Gly GluAsp Trp Glu Asp Tyr Asn Asp 1415 1420 1425 Ala Phe Arg Ala Arg Gln LysGly Lys Ile Tyr Lys Ala Thr Ala 1430 1435 1440 Thr Ser Met Lys Phe TyrPhe Pro Pro Gly Pro Val Ile Glu Pro 1445 1450 1455 Thr Leu Gly Leu Asn1460 <210> SEQ ID NO 31 <211> LENGTH: 254 <212> TYPE: PRT <213>ORGANISM: Arterivirus porcine respiratory and reproductive syndromevirus <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:(1)..(254) <223> OTHER INFORMATION: GP3 (ORF 3), nucleotides 12680 to13444 of the viral sequence <400> SEQUENCE: 31 Met Ala Asn Ser Cys ThrPhe Leu His Ile Phe Leu Cys Cys Ser Phe 1 5 10 15 Leu Tyr Ser Phe CysCys Ala Val Val Ala Gly Ser Asn Ala Thr Tyr 20 25 30 Cys Phe Trp Phe ProLeu Val Arg Gly Asn Phe Ser Phe Glu Leu Met 35 40 45 Val Asn Tyr Thr ValCys Pro Pro Cys Leu Thr Arg Gln Ala Ala Ala 50 55 60 Glu Val Leu Glu ProGly Arg Ser Leu Trp Cys Arg Ile Gly His Asp 65 70 75 80 Arg Cys Gly GluAsp Asp His Asp Glu Leu Gly Phe Met Val Pro Pro 85 90 95 Gly Leu Ser SerGlu Ser His Leu Thr Ser Val Tyr Ala Trp Leu Ala 100 105 110 Phe Leu SerPhe Ser Tyr Thr Ala Gln Phe His Pro Glu Ile Phe Gly 115 120 125 Ile GlyAsn Val Ser Glu Val Tyr Val Asp Ile Lys His Gln Phe Ile 130 135 140 CysAla Val His Asp Gly Gln Asn Thr Thr Leu Pro Arg His Asp Asn 145 150 155160 Ile Ser Ala Val Phe Gln Thr Tyr Tyr Gln His Gln Val Asp Gly Gly 165170 175 Asn Trp Phe His Leu Glu Trp Leu Arg Pro Phe Phe Ser Ser Trp Leu180 185 190 Val Leu Asn Val Ser Trp Phe Leu Arg Arg Ser Pro Ala Ser HisVal 195 200 205 Ser Val Arg Val Phe Gln Thr Ser Lys Pro Thr Leu Pro GlnHis Gln 210 215 220 Ala Leu Leu Ser Ser Arg Thr Ser Ala Ala Leu Gly MetAla Thr Arg 225 230 235 240 Pro Phe Arg Arg Phe Ala Lys Ala Leu Asn AlaAla Arg Arg 245 250 <210> SEQ ID NO 32 <211> LENGTH: 174 <212> TYPE: PRT<213> ORGANISM: Arterivirus porcine respiratory and reproductivesyndrome virus <220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>LOCATION: (1)..(174) <223> OTHER INFORMATION: Protein M (ORF 6),nucleotides 14359 to 14883 of the viral sequence <400> SEQUENCE: 32 MetGly Ser Ser Leu Asp Asp Phe Cys His Asp Ser Thr Ala Pro Gln 1 5 10 15Lys Val Leu Leu Ala Phe Ser Ile Thr Tyr Thr Pro Val Met Ile Tyr 20 25 30Ala Leu Lys Val Ser Arg Gly Arg Leu Leu Gly Leu Leu His Leu Leu 35 40 45Ile Phe Leu Asn Cys Ala Phe Thr Phe Gly Tyr Met Thr Phe Glu His 50 55 60Phe Gln Ser Thr Asn Arg Val Ala Leu Thr Met Gly Ala Val Val Ala 65 70 7580 Leu Leu Trp Gly Val Tyr Ser Ala Ile Glu Thr Trp Lys Phe Ile Thr 85 9095 Ser Arg Cys Arg Leu Cys Leu Leu Gly Arg Lys Tyr Ile Leu Ala Pro 100105 110 Ala His His Val Glu Ser Ala Ala Gly Phe His Pro Ile Ala Ala Asn115 120 125 Asp Asn His Ala Phe Val Val Arg Arg Pro Gly Ser Thr Thr ValAsn 130 135 140 Gly Thr Leu Val Pro Gly Leu Lys Ser Leu Val Leu Gly GlyArg Lys 145 150 155 160 Ala Val Lys Gln Gly Val Val Asn Leu Val Lys TyrAla Lys 165 170

1-20. (canceled).
 21. An isolated nucleic acid comprising a DNA sequenceencoding an infectious RNA molecule encoding a North American PRRSvirus, wherein said DNA sequence is SEQ ID NO:24 or a sequence thathybridizes to the complement of SEQ ID NO:24 under conditions comprisinghybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.
 22. A transfected hostcell comprising a DNA sequence encoding an infectious RNA moleculeencoding a North American PRRS virus, wherein said DNA sequence is SEQID NO:24 or a sequence that hybridizes to the complement of SEQ ID NO:24under conditions comprising hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C., which transfected host cell is capable of expressing the encodedNorth American PRRS virus.
 23. An isolated nucleic acid comprising a DNAsequence encoding an infectious RNA molecule encoding a North AmericanPRRS virus, wherein said DNA sequence is SEQ ID NO:
 24. 24. An isolatednucleic acid in the form of a plasmid, wherein said isolated nucleicacid comprises a DNA sequence encoding an infectious RNA moleculeencoding a North American PRRS virus, wherein said DNA sequence is SEQID NO:
 24. 25. An isolated infectious RNA molecule encoded by anisolated nucleic acid comprising SEQ ID NO: 24, which infectious RNAmolecule encodes a North American PRRS virus.
 26. A recombinant NorthAmerican PRRS virus encoded by an isolated nucleic acid comprising a DNAsequence encoding an infectious RNA molecule encoding a North AmericanPRRS virus, wherein said DNA sequence is SEQ ID NO: 24.